Anti-PD-1 Antibodies and Uses Thereof

ABSTRACT

Provided herein are various embodiments relating to antibodies. Some of the embodiments include antagonist antibodies that bind PD-1. Such antibodies can be used in methods to treat, for example, cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/845,805, filed Apr. 10, 2020, which is a divisional of U.S.application Ser. No. 15/800,346, filed Nov. 1, 2017, now U.S. Pat. No.10,654,929, which claims the benefit of priority of U.S. ProvisionalApplication No. 62/416,602, filed Nov. 2, 2016, the contents of each ofwhich are incorporated by reference herein in their entirety for anypurpose.

FIELD OF THE INVENTION

Antibodies that bind to Programmed Death-1 protein (PD-1) are provided.Methods of treatment comprising administering anti-PD-1 antibodies arealso provided.

BACKGROUND

The Programmed Death 1 (PD-1) protein is an inhibitory member of theCD28 family of receptors, which also includes CD28, CTLA-4, ICOS andBTLA. PD-1 is expressed on the surface of activated B cells, T cells,and myeloid cells. PD-1 contains a membrane proximal immunoreceptortyrosine inhibitory motif (ITIM) and a membrane distal tyrosine-basedswitch motif (ITSM). Although structurally similar to CTLA-4, PD-1 lacksthe MYPPY motif that is critical for B7-1 and B7-2 binding. In addition,although CD28, ICOS and CTLA-4 (other members of the CD28 family) allhave an unpaired cysteine residue allowing for homodimerization, PD-1 isbelieved to exist as a monomer, lacking the unpaired cysteine residuecharacteristic in other CD28 family members. The PD-1 receptor has twoligands, PD-ligand-1 (PD-L1) and PD-L2. The term “PD-L1” refers to theligand of the PD-1 receptor also known as CD274 and B7H 1. PD-L1 is a290 amino acid protein with an extracellular IgV-like domain, anextracellular IgC-like domain, a transmembrane domain and a highlyconserved intracellular domain of approximately 30 amino acids. PD-L1 isconstitutively expressed on many cells such as antigen presenting cells(e.g., dendritic cells, macrophages, and B-cells) and on hematopoieticand non-hematopoietic cells (e.g., vascular endothelial cells,pancreatic islets, and sites of immune privilege). The term “PD-L2”refers to the ligand of the PD-1 receptor also known as CD273 and B7-DC.PD-L2 has an extracellular IgV-like domain, an extracellular IgC-likedomain, a transmembrane domain and an intracellular domain ofapproximately 30 amino acids in humans. PD-L2 has a more restrictedexpression than PD-L1, with its expression largely confined tohematopoietic cells including macrophages, dendritic cells, some B cellsubsets and bone marrow-derived mast cells.

PD-1 functions as an immune checkpoint and works to prevent theactivation of T-cells. PD-1 antagonists activate the immune system toattack tumors and have shown success in treating cancers, and in someinstances, with less toxicity than other chemotherapeutic treatments.PD-1 antagonists can also be used in combination regimens with otherchemotherapeutic agents. Currently approved PD-1 antagonists includeanti-PD-1 antibodies, Opdivo® and Keytruda®, and anti-PD-L1 antibody,Tecentriq™.

There remains a need for additional antagonists of PD-1 for treatment ofcancer and other diseases and disorders.

SUMMARY

Antibodies that bind Programmed Death 1 (PD-1) are provided, wherein theantibodies bind human PD-1 and mouse PD-1, and wherein the antibodiesblock binding of PD-L1 and/or PD-L2 to PD-1.

In some embodiments, an isolated antibody that binds to Programmed Death1 (PD-1) is provided, wherein the antibody binds to an epitopecomprising amino acids 126 to 136 of human PD-1. In some embodiments,amino acids 126 to 136 are numbered according to SEQ ID NO: 1. In someembodiments, the antibody binds to human PD-1 of SEQ ID NO: 382 with atleast 10-fold greater affinity than the antibody binds to one or morePD-1 variants selected from I126A, L128A, A132L, I134A, and E136A.

In some embodiments, the antibody:

-   -   a) binds to human PD-1 ECD-Fc of SEQ ID NO: 401 with at least        10-fold greater affinity than the antibody binds to PD-1 variant        I126A ECD-Fc (SEQ ID NO: 389); and/or    -   b) binds to human PD-1 ECD-Fc of SEQ ID NO: 401 with at least        10-fold greater affinity than the antibody binds to PD-1 variant        L128A ECD-Fc (SEQ ID NO: 390); and/or    -   c) binds to human PD-1 ECD-Fc of SEQ ID NO: 401 with at least        10-fold greater affinity than the antibody binds to PD-1 variant        A132L ECD-Fc (SEQ ID NO: 391); and/or    -   d) binds to human PD-1 ECD-Fc of SEQ ID NO: 401 with at least        10-fold greater affinity than the antibody binds to PD-1 variant        I134A ECD-Fc (SEQ ID NO: 392); and/or    -   e) binds to human PD-1 ECD-Fc of SEQ ID NO: 401 with at least        10-fold greater affinity than the antibody binds to PD-1 variant        E136A ECD-Fc (SEQ ID NO: 393).

In some embodiments, the antibody binds to mouse PD-1 ECD-Fc of SEQ IDNO: 403. In some embodiments, the antibody binds to mouse PD-1 ECD-Fc ofSEQ ID NO: 403 with at least 10-fold greater affinity than the antibodybinds to mouse PD-1 variant H129P ECD-Fc (SEQ ID NO: 394). In someembodiments, the antibody binds to rat PD-1 variant P129H ECD-Fc (SEQ IDNO: 397) with at least 10-fold greater affinity than the antibody bindsto rat PD-1 ECD-Fc (SEQ ID NO: 405).

In various embodiments, affinity is determined by biolayerinterferometry.

In some embodiments, the antibody comprises (a) HCDR1 comprising theamino acid sequence of SEQ ID NO: 21; (b) HCDR2 comprising the aminoacid sequence of SEQ ID NO: 22; (c) HCDR3 comprising the amino acidsequence of SEQ ID NO: 23; (d) LCDR1 comprising the amino acid sequenceof SEQ ID NO: 25; (e) LCDR2 comprising the amino acid sequence of SEQ IDNO: 26; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO:27. In some embodiments, the antibody comprises a heavy chain variableregion (V_(H)) that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20and a light chain variable region (V_(L)) that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the aminoacid sequence of SEQ ID NO: 24. In some embodiments, the antibodycomprises a V_(H) comprising the amino acid sequence of SEQ ID NO: 20and a V_(L) comprising the amino acid sequence of SEQ ID NO: 24.

In some embodiments, an antibody that binds to Programmed Death 1 (PD-1)is provided, wherein the antibody comprises:

-   -   i) (a) HCDR1 comprising the amino acid sequence of SEQ ID NO:        5; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO:        6; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO:        7; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO:        9; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO:        10; and (f) LCDR3 comprising the amino acid sequence of SEQ ID        NO: 11; or    -   ii) (a) HCDR1 comprising the amino acid sequence of SEQ ID NO:        13; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO:        14; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO:        15; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO:        17; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO:        18; and (f) LCDR3 comprising the amino acid sequence of SEQ ID        NO: 19; or    -   iii) (a) HCDR1 comprising the amino acid sequence of SEQ ID NO:        21; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO:        22; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO:        23; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO:        25; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO:        26; and (f) LCDR3 comprising the amino acid sequence of SEQ ID        NO: 27; or    -   iv) (a) HCDR1 comprising the amino acid sequence of SEQ ID NO:        29; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO:        30; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO:        31; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO:        33; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO:        34; and (f) LCDR3 comprising the amino acid sequence of SEQ ID        NO: 35; or    -   v) (a) HCDR1 comprising the amino acid sequence of SEQ ID NO:        37; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO:        38; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO:        39; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO:        41; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO:        42; and (f) LCDR3 comprising the amino acid sequence of SEQ ID        NO: 43; or    -   vi) (a) HCDR1 comprising an amino acid sequence selected from        SEQ ID NOs: 45, 53, 61, 69, 77, 85, 93, 101, 109 and 117; (b)        HCDR2 comprising an amino acid sequence selected from SEQ ID        NOs: 46, 54, 62, 70, 78, 86, 94, 102, 110 and 118; (c) HCDR3        comprising an amino acid sequence selected from SEQ ID NOs: 47,        55, 63, 71, 79, 87, 95, 103, 111, and 119; (d) LCDR1 comprising        an amino acid sequence selected from SEQ ID NOs: 49, 57, 65, 73,        81, 89, 97, 105, 113, and 121; (e) LCDR2 comprising an amino        acid sequence selected from SEQ ID NOs: 50, 58, 66, 74, 82, 90,        98, 106, 114, and 122; and (f) LCDR3 comprising an amino acid        sequence selected from SEQ ID NOs: 51, 59, 67, 75, 83, 91, 99,        107, 115, and 123; or    -   vii)(a) HCDR1 comprising an amino acid sequence selected from        SEQ ID NOs: 125, 133, 141, 149, 157, 165, 173, 181, 189, 197,        205, 213, 221, 229, 237, 245, 253, 261, 269, 277 and 285; (b)        HCDR2 comprising an amino acid sequence selected from SEQ ID        NOs: 126, 134, 142, 150, 158, 166, 174, 182, 190, 198, 206, 214,        222, 230, 238, 246, 254, 262, 270, 278 and 286; (c) HCDR3        comprising an amino acid sequence selected from SEQ ID NOs: 127,        135, 143, 151, 159, 167, 175, 183, 191, 199, 207, 215, 223, 231,        239, 247, 255, 263, 271, 279 and 287; (d) LCDR1 comprising an        amino acid sequence selected from SEQ ID NOs: 129, 137, 145,        153, 161, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249,        257, 265, 273, 281 and 289; (e) LCDR2 comprising an amino acid        sequence selected from SEQ ID NOs: 130, 138, 146, 154, 162, 170,        178, 186, 194, 202, 210, 218, 226, 234, 242, 250, 258, 266, 274,        282, and 290; and (f) LCDR3 comprising an amino acid sequence        selected from SEQ ID NOs: 131, 139, 147, 155, 163, 171, 179,        187, 195, 203, 211, 219, 227, 235, 243, 251, 259, 267, 275, 283        and 291; or    -   viii) (a) HCDR1 comprising an amino acid sequence selected from        SEQ ID NOs: 293, 301, 309 and 317; (b) HCDR2 comprising an amino        acid sequence selected from SEQ ID NOs: 294, 302, 310 and        318; (c) HCDR3 comprising an amino acid sequence selected from        SEQ ID NOs: 295, 303, 311 and 319; (d) LCDR1 comprising an amino        acid sequence selected from SEQ ID NOs: 297, 305, 313, and        321; (e) LCDR2 comprising an amino acid sequence selected from        SEQ ID NOs: 298, 306, 314 and 322; and (f) LCDR3 comprising an        amino acid sequence selected from SEQ ID NOs: 299, 307, 315, and        323.

In some embodiments, an antibody that binds PD-1 is provided, whichcomprises a heavy chain variable region (V_(H)) and a light chainvariable region (V_(L)), wherein:

-   -   i) the V_(H) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,        98%, 99%, or 100% identical to the amino acid sequence of SEQ ID        NO: 4 and the V_(L) is at least 90%, 91%, 92%, 93%, 94%, 95%,        96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence        of SEQ ID NO: 8; or    -   ii) the V_(H) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, 99%, or 100% identical to the amino acid sequence of        SEQ ID NO: 12 and the V_(L) is at least 90%, 91%, 92%, 93%, 94%,        95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid        sequence of SEQ ID NO: 16; or    -   iii) the V_(H) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, 99%, or 100% identical to the amino acid sequence of        SEQ ID NO: 20 and the V_(L) is at least 90%, 91%, 92%, 93%, 94%,        95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid        sequence of SEQ ID NO: 24; or    -   iv) the V_(H) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, 99%, or 100% identical to the amino acid sequence of        SEQ ID NO: 28 and the V_(L) is at least 90%, 91%, 92%, 93%, 94%,        95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid        sequence of SEQ ID NO: 32; or    -   v) the V_(H) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,        98%, 99%, or 100% identical to the amino acid sequence of SEQ ID        NO: 36 and the V_(L) is at least 90%, 91%, 92%, 93%, 94%, 95%,        96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence        of SEQ ID NO: 40; or    -   vi) the V_(H) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, 99%, or 100% identical to an amino acid sequence        selected from SEQ ID NOs: 44, 52, 60, 68, 76, 84, 92, 100, 108        and 116; and the V_(L) is at least 90%, 91%, 92%, 93%, 94%, 95%,        96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence        selected from SEQ ID NOs: 48, 56, 64, 72, 80, 88, 96, 104, 112,        and 120; or    -   vii) the V_(H) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, 99%, or 100% identical to an amino acid sequence        selected from SEQ ID NOs: 124, 132, 140, 148, 156, 164, 172,        180, 188, 196, 204, 212, 220, 228, 236, 244, 252, 260, 268, 276,        and 284; and the V_(L) is at least 90%, 91%, 92%, 93%, 94%, 95%,        96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence        selected from SEQ ID NOs: 128, 136, 144, 152, 160, 168, 176,        184, 192, 200, 208, 216, 224, 232, 240, 248, 256, 264, 272, 280        and 288; or    -   viii) the V_(H) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, 99%, or 100% identical to an amino acid sequence        selected from SEQ ID NOs: 292, 300, 308 and 316; and the V_(L)        is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or        100% identical to an amino acid sequence selected from SEQ ID        NOs: 296, 304, 312, and 320.

In some embodiments, an antibody that binds PD-1 is provided, whichcomprises a heavy chain variable region (V_(H)) and a light chainvariable region (V_(L)), wherein:

-   -   i) the V_(H) comprises the amino acid sequence of SEQ ID NO: 4        and the V_(L) comprises the amino acid sequence of SEQ ID NO: 8;        or    -   ii) the V_(H) comprises the amino acid sequence of SEQ ID NO: 12        and the V_(L) comprises the amino acid sequence of SEQ ID NO:        16; or    -   iii) the V_(H) comprises the amino acid sequence of SEQ ID NO:        20 and the V_(L) comprises the amino acid sequence of SEQ ID NO:        24; or    -   iv) the V_(H) comprises the amino acid sequence of SEQ ID NO: 28        and the V_(L) comprises the amino acid sequence of SEQ ID NO:        32; or    -   v) the V_(H) comprises the amino acid sequence of SEQ ID NO: 36        and the V_(L) comprises the amino acid sequence of SEQ ID NO:        40; or    -   vi) the V_(H) comprises the amino acid sequence of SEQ ID NO: 44        and the V_(L) comprises the amino acid sequence of SEQ ID NO:        48; or    -   vii) the V_(H) comprises the amino acid sequence of SEQ ID NO:        52 and the V_(L) comprises the amino acid sequence of SEQ ID NO:        56; or    -   viii) the V_(H) comprises the amino acid sequence of SEQ ID NO:        60 and the V_(L) comprises the amino acid sequence of SEQ ID NO:        64; or    -   ix) the V_(H) comprises the amino acid sequence of SEQ ID NO: 68        and the V_(L) comprises the amino acid sequence of SEQ ID NO:        72; or    -   x) the V_(H) comprises the amino acid sequence of SEQ ID NO: 76        and the V_(L) comprises the amino acid sequence of SEQ ID NO:        80; or    -   xi) the V_(H) comprises the amino acid sequence of SEQ ID NO: 84        and the V_(L) comprises the amino acid sequence of SEQ ID NO:        88; or    -   xii) the V_(H) comprises the amino acid sequence of SEQ ID NO:        92 and the V_(L) comprises the amino acid sequence of SEQ ID NO:        96; or    -   xiii) the V_(H) comprises the amino acid sequence of SEQ ID NO:        100 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 104; or    -   xiv) the V_(H) comprises the amino acid sequence of SEQ ID NO:        108 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 112; or    -   xv) the V_(H) comprises the amino acid sequence of SEQ ID NO:        116 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 120; or    -   xvi) the V_(H) comprises the amino acid sequence of SEQ ID NO:        124 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 128; or    -   xvii) the V_(H) comprises the amino acid sequence of SEQ ID NO:        132 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 136; or    -   xviii) the V_(H) comprises the amino acid sequence of SEQ ID NO:        140 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 144; or    -   xix) the V_(H) comprises the amino acid sequence of SEQ ID NO:        148 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 152; or    -   xx) the V_(H) comprises the amino acid sequence of SEQ ID NO:        156 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 160; or    -   xxi) the V_(H) comprises the amino acid sequence of SEQ ID NO:        164 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 168; or    -   xxii) the V_(H) comprises the amino acid sequence of SEQ ID NO:        172 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 176; or    -   xxiii) the V_(H) comprises the amino acid sequence of SEQ ID NO:        180 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 184; or    -   xxiv) the V_(H) comprises the amino acid sequence of SEQ ID NO:        188 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 192; or    -   xxv) the V_(H) comprises the amino acid sequence of SEQ ID NO:        196 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 200; or    -   xxvi) the V_(H) comprises the amino acid sequence of SEQ ID NO:        204 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 208; or    -   xxvii) the V_(H) comprises the amino acid sequence of SEQ ID NO:        212 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 216; or    -   xxviii) the V_(H) comprises the amino acid sequence of SEQ ID        NO: 220 and the V_(L) comprises the amino acid sequence of SEQ        ID NO: 224; or    -   xxix) the V_(H) comprises the amino acid sequence of SEQ ID NO:        228 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 232; or    -   xxx) the V_(H) comprises the amino acid sequence of SEQ ID NO:        236 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 240; or    -   xxxi) the V_(H) comprises the amino acid sequence of SEQ ID NO:        244 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 248; or    -   xxxii) the V_(H) comprises the amino acid sequence of SEQ ID NO:        252 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 256; or    -   xxxiii) the V_(H) comprises the amino acid sequence of SEQ ID        NO: 260 and the V_(L) comprises the amino acid sequence of SEQ        ID NO: 264; or    -   xxxiv) the V_(H) comprises the amino acid sequence of SEQ ID NO:        268 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 272; or    -   xxxv) the V_(H) comprises the amino acid sequence of SEQ ID NO:        276 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 280; or    -   xxxvi) the V_(H) comprises the amino acid sequence of SEQ ID NO:        284 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 288; or    -   xxxvii) the V_(H) comprises the amino acid sequence of SEQ ID        NO: 292 and the V_(L) comprises the amino acid sequence of SEQ        ID NO: 296; or    -   xxxviii) the V_(H) comprises the amino acid sequence of SEQ ID        NO: 300 and the V_(L) comprises the amino acid sequence of SEQ        ID NO: 304; or    -   xxxix) the V_(H) comprises the amino acid sequence of SEQ ID NO:        308 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 312; or    -   xl) the V_(H) comprises the amino acid sequence of SEQ ID NO:        316 and the V_(L) comprises the amino acid sequence of SEQ ID        NO: 320.

In some embodiments, an antibody that competes with an antibody providedherein for binding to human PD-1 is provided, wherein the antibody bindsto human PD-1 and mouse PD-1, and where in the antibody inhibits bindingof human PD-1 to human PD-L1, inhibits binding of human PD-1 to humanPD-L2, inhibits binding of mouse PD-1 to mouse PD-L1, and inhibitsbinding of mouse PD-1 to mouse PD-L2.

In some embodiments, an antibody provided herein binds to human PD-1with an affinity (K_(D)) of less than 5 nM. In some embodiments, anantibody provided herein binds to mouse PD-1 with an affinity (K_(D)) ofless than 10 nM. In some embodiments, affinity is determined usingbiolayer interferometry.

In some embodiments, an antibody provided herein is a monoclonalantibody. In some embodiments, an antibody provided herein is a humanantibody, chimeric antibody, or a humanized antibody. In someembodiments, an antibody provided herein is an antibody fragmentselected from a Fab, Fab′, Fv, scFv or (Fab′)₂ fragment. In someembodiments, an antibody provided herein is a full length antibody. Insome embodiments, an antibody provided herein is an IgG1 or IgG4antibody.

In some embodiments, an antibody provided herein binds human PD-1. Insome embodiments, human PD-1 comprises the amino acid sequence of SEQ IDNO: 382. In some embodiments, an antibody provided herein binds mousePD-1. In some embodiments, mouse PD-1 comprises the amino acid sequenceof SEQ ID NO: 383. In some embodiments, an antibody provided hereinbinds cynomolgus monkey PD-1. In some embodiments, cynomolgus monkeyPD-1 comprises the amino acid sequence of SEQ ID NO: 384.

In some embodiments, an antibody provided herein inhibits binding ofPD-1 to PD-L1. In some embodiments, an antibody provided herein inhibitsbinding of PD-1 to PD-L2. In some embodiments, an antibody providedherein inhibits binding of PD-1 to PD-L1 and inhibits the binding ofPD-1 to PD-L2.

In some embodiments, administration of an antibody provided herein to amammal increases the level of at least one cytokine selected from IFNγand IL-2. In some embodiments, an antibody provided herein increases thelevel of at least one cytokine selected from IFNγ and IL-2 by at least2-fold. In some embodiments, the level of the cytokine is measured 1hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 36hours, or 48 hours after administration of the antibody. In someembodiments, at least one cytokine is IFNγ. In some embodiments, atleast one cytokine is IL-2. In some embodiments, the level of thechemokine is measured 24 hours after administration of the antibody.

In some embodiments, administration of an antibody provided herein to amammal enhances an immune response in the mammal. In some embodiments,administration of an antibody provided herein to a mammal results inactivation of T cells in the mammal. In some embodiments, administrationof an antibody provided herein to a mammal reduces tumor size in amammal with cancer. In some embodiments, the mammal is a human. In someembodiments, the human has cancer. In some embodiments, the cancer isselected from melanoma, non-small cell lung cancer (NSCLC), renal cellcarcinoma (RCC), gastric cancer, bladder cancer, diffuse large B-celllymphoma (DLBCL), Hodgkin's lymphoma, ovarian cancer, head & necksquamous cell cancer (HNSCC), mesothelioma, and triple negative breastcancer (TNBC). In some embodiments, the cancer is selected frommelanoma, gastric cancer, head & neck squamous cell cancer (HNSCC),non-small cell lung cancer (NSCLC), and triple negative breast cancer(TNBC).

In some embodiments, an isolated nucleic acid is provided, which encodesan antibody provided herein. In some embodiments, a vector is provided,which comprises the nucleic acid. In some embodiments, a host cell isprovided, which comprises the vector. In some embodiments, a host cellis provided, which produces an antibody provided herein. In someembodiments, a method for making an anti-PD-1 antibody is provided,comprising culturing the host cell under conditions suitable forexpression of the antibody. In some embodiments, the method furthercomprises recovering the antibody produced by the host cell.

In some embodiments, a pharmaceutical composition is provided,comprising an anti-PD-1 antibody provided herein and a pharmaceuticallyacceptable carrier.

In some embodiments, a method of treating cancer in a mammal isprovided, comprising administering an effective amount of an anti-PD-1antibody provided herein, or a pharmaceutical composition comprising ananti-PD-1 antibody provided herein and a pharmaceutically acceptablecarrier. In some embodiments, the cancer is selected from melanoma,non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), gastriccancer, bladder cancer, diffuse large B-cell lymphoma (DLBCL), Hodgkin'slymphoma, ovarian cancer, head & neck squamous cell cancer (HNSCC),mesothelioma, and triple negative breast cancer (TNBC). In someembodiments, the cancer is selected from melanoma, gastric cancer, head& neck squamous cell cancer (HNSCC), non-small cell lung cancer (NSCLC),and triple negative breast cancer (TNBC).

In some embodiments, a method of enhancing an immune response in amammal is provided, comprising administering an effective amount of ananti-PD-1 antibody provided herein, or a pharmaceutical compositioncomprising an anti-PD-1 antibody provided herein and a pharmaceuticallyacceptable carrier.

In some embodiments, a method of increasing activation of a T cell in amammal is provided, comprising administering an effective amount of ananti-PD-1 antibody provided herein, or a pharmaceutical compositioncomprising an anti-PD-1 antibody provided herein and a pharmaceuticallyacceptable carrier.

In some embodiments, a method of reducing tumor size in a in a mammalwith cancer is provided, comprising administering an effective amount ofan anti-PD-1 antibody provided herein, or a pharmaceutical compositioncomprising an anti-PD-1 antibody provided herein and a pharmaceuticallyacceptable carrier.

In any of the embodiments provided herein, the mammal may be a human.

In some embodiments, the mammal is administered at least one additionaltherapeutic agent. In some such embodiments, the additional therapeuticagent is administered concurrently or sequentially with the anti-PD-1antibody. In some embodiments, the additional therapeutic agent isselected from an anti-ICOS antibody and an anti-CTLA4 antibody. In someembodiments, the additional therapeutic agent is an anti-ICOS antibody.In some embodiments, the additional therapeutic agent is a cancervaccine. In some embodiments, the cancer vaccine is selected from a DNAvaccine, an engineered virus vaccine, an engineered tumor cell vaccine,and a cancer vaccine developed using neoantigens.

In some embodiments, use of an antibody provided herein is provided forthe manufacture of a medicament to treat cancer. In some embodiments,the cancer is selected from melanoma, non-small cell lung cancer(NSCLC), renal cell carcinoma (RCC), gastric cancer, bladder cancer,diffuse large B-cell lymphoma (DLBCL), Hodgkin's lymphoma, ovariancancer, head & neck squamous cell cancer (HNSCC), mesothelioma, andtriple negative breast cancer (TNBC). In some embodiments, the cancer isselected from melanoma, gastric cancer, head & neck squamous cell cancer(HNSCC), non-small cell lung cancer (NSCLC), and triple negative breastcancer (TNBC). In some embodiments, the medicament is for administrationwith at least one additional therapeutic agent. In some embodiments, theadditional therapeutic agent is selected from an anti-ICOS antibody andan anti-CTLA4 antibody. In some embodiments, the additional therapeuticagent is an anti-ICOS antibody. In some embodiments, the additionaltherapeutic agent is a cancer vaccine. In some embodiments, the cancervaccine is selected from a DNA vaccine, an engineered virus vaccine, anengineered tumor cell vaccine, and a cancer vaccine developed usingneoantigens.

In some embodiments, the present disclosure provides uses of an antibodyprovided herein or a pharmaceutical composition comprising an antibodyprovided herein and a pharmaceutically acceptable carrier for treatingcancer. In some embodiments, the cancer is selected from melanoma,non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), gastriccancer, bladder cancer, diffuse large B-cell lymphoma (DLBCL), Hodgkin'slymphoma, ovarian cancer, head & neck squamous cell cancer (HNSCC),mesothelioma, and triple negative breast cancer (TNBC). In someembodiments, the cancer is selected from melanoma, gastric cancer, head& neck squamous cell cancer (HNSCC), non-small cell lung cancer (NSCLC),and triple negative breast cancer (TNBC).

In some embodiments, the present disclosure provides uses of an antibodyprovided herein or a pharmaceutical composition comprising an antibodyprovided herein and a pharmaceutically acceptable carrier, and at leastone additional therapeutic agent, for treating cancer. In someembodiments, the additional therapeutic agent is selected from ananti-ICOS antibody and an anti-CTLA4 antibody. In some embodiments, theadditional therapeutic agent is an anti-ICOS antibody. In someembodiments, the additional therapeutic agent is a cancer vaccine. Insome embodiments, the cancer vaccine is selected from a DNA vaccine, anengineered virus vaccine, an engineered tumor cell vaccine, and a cancervaccine developed using neoantigens. In some embodiments, the cancer isselected from melanoma, non-small cell lung cancer (NSCLC), renal cellcarcinoma (RCC), gastric cancer, bladder cancer, diffuse large B-celllymphoma (DLBCL), Hodgkin's lymphoma, ovarian cancer, head & necksquamous cell cancer (HNSCC), mesothelioma, and triple negative breastcancer (TNBC). In some embodiments, the cancer is selected frommelanoma, gastric cancer, head & neck squamous cell cancer (HNSCC),non-small cell lung cancer (NSCLC), and triple negative breast cancer(TNBC).

In some embodiments, an antibody provided herein or a pharmaceuticalcomposition comprising an antibody provided herein and apharmaceutically acceptable carrier for use in treating cancer isprovided. In some embodiments, the cancer is selected from melanoma,non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), gastriccancer, bladder cancer, diffuse large B-cell lymphoma (DLBCL), Hodgkin'slymphoma, ovarian cancer, head & neck squamous cell cancer (HNSCC),mesothelioma, and triple negative breast cancer (TNBC). In someembodiments, the cancer is selected from melanoma, gastric cancer, head& neck squamous cell cancer (HNSCC), non-small cell lung cancer (NSCLC),and triple negative breast cancer (TNBC).

In some embodiments, a method of increasing the level of at least onecytokine selected from IFNγ and IL-2 in a mammal is provided, comprisingadministering to the mammal an antibody provided herein. In someembodiments, the level of the cytokine is measured 1 hour, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours,16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 36 hours, or 48 hoursafter administration of the antibody. In some embodiments, at least onecytokine is IFNγ. In some embodiments, at least one cytokine is IL-2. Insome embodiments, the level of the cytokine is measured 24 hours afteradministration of the antibody. In some embodiments, the mammal is ahuman. In some embodiments, the human has cancer. In some embodiments,the cancer is selected from melanoma, non-small cell lung cancer(NSCLC), renal cell carcinoma (RCC), gastric cancer, bladder cancer,diffuse large B-cell lymphoma (DLBCL), Hodgkin's lymphoma, ovariancancer, head & neck squamous cell cancer (HNSCC), mesothelioma, andtriple negative breast cancer (TNBC). In some embodiments, the cancer isselected from melanoma, gastric cancer, head & neck squamous cell cancer(HNSCC), non-small cell lung cancer (NSCLC), and triple negative breastcancer (TNBC).

In some embodiments, an immune response is enhanced followingadministration of the anti-PD-1 antibody. In some embodiments,activation of T cells is increased following administration of theanti-PD-1 antibody. In some embodiments, tumor size is decreasedfollowing administration of the anti-PD-1 antibody.

In some embodiments, the mammal is administered at least one additionaltherapeutic agent. In some embodiments, the additional therapeutic agentis administered concurrently or sequentially with the anti-PD-1antibody. In some embodiments, the additional therapeutic agent isselected from an anti-ICOS antibody and an anti-CTLA4 antibody. In someembodiments, the additional therapeutic is an anti-ICOS antibody. Insome embodiments, the additional therapeutic agent is a cancer vaccine.In some embodiments, the cancer vaccine is selected from a DNA vaccine,an engineered virus vaccine, an engineered tumor cell vaccine, and acancer vaccine developed using neoantigens.

In some embodiments, a sample of the cancer from the mammal has beendetermined to express PD-1. In some embodiments, the sample shows 1+,2+, or 3+ staining of PD-1 by immunohistochemistry (IHC). In someembodiments, the sample has been determined to have an elevated level ofPD-L1. In some embodiments, PD-L1 levels are determined using IHC.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows IFNγ concentrations after mouse splenocyte T cellsactivated with anti-CD3 and anti-CD28 antibodies are contacted withanti-PD-1 antibodies. Mouse IgG1 and rat IgG2a were used as controls.RMP1-14 was used as a positive control anti-PD-1 antibody.

FIG. 2 shows the EC₅₀ of increase in IL-2 in whole blood samplesactivated with Staphylococcal enterotoxin B (SEB) after being incubatedwith anti-PD1 antibodies. nivolumab (Nivo) and pembrolizumab (Pembro)were used as control anti-PD1 antibodies. Effects from antibodies 12228(28), 13406 (6), 13407 (7), 13408 (8), and 13409 (9) are shown.

FIG. 3A-3B show the ability of anti-PD-1 antibodies disclosed herein toblock binding of mouse ligands to mouse PD-1. Octet analysis on aForte-Bio instrument was used to assess the effect of the antibodies onthe interaction between mouse PD-L1 and mouse PD-L2 with mouse PD-1.

FIGS. 4A-1, 4A-2, 4B-1, 4B-2, and 4C show the ability of anti-PD-1antibodies disclosed herein to block the binding of human ligands tohuman PD-1. Octet analysis on a Forte-Bio instrument was used to assessthe effect of the antibodies on the interaction between human PD-L1 andhuman PD-L2 with human PD-1.

FIG. 5 shows the reduction in tumor sizes in mouse tumor models uponadministration of an anti-PD-1 antibody disclosed herein. C57BL6/J micewere injected subcutaneously in their flanks with MC38 cells. Mouse IgG1(MOPC-21) and rat IgG2a (2A3) were used as controls. RMP1-14 was used asa positive control anti-PD1 antibody.

DETAILED DESCRIPTION OF SOME EMBODIMENTS

Antibodies that bind PD-1 are provided. Antibody heavy chains and lightchains that are capable of forming antibodies that bind PD-1 are alsoprovided. In addition, antibodies, heavy chains, and light chainscomprising one or more particular complementarity determining regions(CDRs) are provided. Polynucleotides encoding antibodies to PD-1 areprovided. Polynucleotides encoding antibody heavy chains or lightschains are also provided. Methods of producing and/or purifyingantibodies to PD-1 are provided. Methods of treatment using antibodiesto PD-1 are provided. Such methods include, but are not limited to,methods of treating cancer. Methods of detecting PD-1 are provided. Suchmethods include methods to identify an individual who may benefit fromtreatment with an anti-PD-1 antibody, to monitor treatment of anindividual with an anti-PD-1 antibody and to improve therapeuticefficacy of an anti-PD-1 antibody in an individual.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

All references cited herein, including patent applications, patentpublications, and Genbank Accession numbers are herein incorporated byreference, as if each individual reference were specifically andindividually indicated to be incorporated by reference in its entirety.

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3rd. edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS INMOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)); the seriesMETHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICALAPPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMALCELL CULTURE (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J. B. LippincottCompany, 1993); and updated versions thereof.

I. Definitions

Unless otherwise defined, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context or expressly indicated, singularterms shall include pluralities and plural terms shall include thesingular. For any conflict in definitions between various sources orreferences, the definition provided herein will control.

It is understood that embodiments of the invention described hereininclude “consisting” and/or “consisting essentially of” embodiments. Asused herein, the singular form “a”, “an”, and “the” includes pluralreferences unless indicated otherwise. Use of the term “or” herein isnot meant to imply that alternatives are mutually exclusive.

In this application, the use of “or” means “and/or” unless expresslystated or understood by one skilled in the art. In the context of amultiple dependent claim, the use of “or” refers back to more than onepreceding independent or dependent claim.

As is understood by one skilled in the art, reference to “about” a valueor parameter herein includes (and describes) embodiments that aredirected to that value or parameter per se. For example, descriptionreferring to “about X” includes description of “X”.

The terms “nucleic acid molecule”, “nucleic acid” and “polynucleotide”may be used interchangeably, and refer to a polymer of nucleotides. Suchpolymers of nucleotides may contain natural and/or non-naturalnucleotides, and include, but are not limited to, DNA, RNA, and PNA.“Nucleic acid sequence” refers to the linear sequence of nucleotidesthat comprise the nucleic acid molecule or polynucleotide.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, and are not limited to a minimumlength. Such polymers of amino acid residues may contain natural ornon-natural amino acid residues, and include, but are not limited to,peptides, oligopeptides, dimers, trimers, and multimers of amino acidresidues. Both full-length proteins and fragments thereof areencompassed by the definition. The terms also include post-expressionmodifications of the polypeptide, for example, glycosylation,sialylation, acetylation, phosphorylation, and the like. Furthermore,for purposes of the present disclosure, a “polypeptide” refers to aprotein which includes modifications, such as deletions, additions, andsubstitutions (generally conservative in nature), to the nativesequence, as long as the protein maintains the desired activity. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe proteins or errors due to PCR amplification.

“PD-1” and “programmed death 1” as used herein refer to any native PD-1that results from expression and processing of PD-1 in a cell. The termincludes PD-1 from any vertebrate source, including mammals such asprimates (e.g., humans and cynomolgus monkeys) and rodents (e.g., miceand rats), unless otherwise indicated. The term also includes naturallyoccurring variants of PD-1, e.g., splice variants or allelic variants.The amino acid sequence of an exemplary human PD-1 precursor protein(with signal sequence, amino acids 1-20) is shown in SEQ ID NO: 1. Theamino acid sequence of an exemplary mature human PD-1 is shown in SEQ IDNO: 382. The amino acid sequence of an exemplary mouse PD-1 precursorprotein (with signal sequence, amino acids 1-20) is shown in SEQ ID NO:2. The amino acid sequence of an exemplary mature mouse PD-1 is shown inSEQ ID NO: 383. The amino acid sequence of an exemplary cynomolgusmonkey PD-1 precursor protein (with signal sequence, amino acids 1-20)is shown in SEQ ID NO: 3. The amino acid sequence of an exemplary maturecynomolgus monkey PD-1 is shown in SEQ ID NO: 384.

The term “specifically binds” to an antigen or epitope is a term that iswell understood in the art, and methods to determine such specificbinding are also well known in the art. A molecule is said to exhibit“specific binding” or “preferential binding” if it reacts or associatesmore frequently, more rapidly, with greater duration and/or with greateraffinity with a particular cell or substance than it does withalternative cells or substances. An antibody “specifically binds” or“preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. For example, an antibody that specifically orpreferentially binds to an PD-1 epitope is an antibody that binds thisepitope with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other PD-1 epitopes or non-PD-1epitopes. It is also understood by reading this definition that, forexample, an antibody (or moiety or epitope) that specifically orpreferentially binds to a first target may or may not specifically orpreferentially bind to a second target. As such, “specific binding” or“preferential binding” does not necessarily require (although it caninclude) exclusive binding. Generally, but not necessarily, reference tobinding means preferential binding. “Specificity” refers to the abilityof a binding protein to selectively bind an antigen.

As used herein, “substantially pure” refers to material which is atleast 50% pure (that is, free from contaminants), more preferably, atleast 90% pure, more preferably, at least 95% pure, yet more preferably,at least 98% pure, and most preferably, at least 99% pure.

As used herein, the term “epitope” refers to a site on a target molecule(for example, an antigen, such as a protein, nucleic acid, carbohydrateor lipid) to which an antigen-binding molecule (for example, anantibody, antibody fragment, or scaffold protein containing antibodybinding regions) binds. Epitopes often include a chemically activesurface grouping of molecules such as amino acids, polypeptides or sugarside chains and have specific three-dimensional structuralcharacteristics as well as specific charge characteristics. Epitopes canbe formed both from contiguous and/or juxtaposed noncontiguous residues(for example, amino acids, nucleotides, sugars, lipid moiety) of thetarget molecule. Epitopes formed from contiguous residues (for example,amino acids, nucleotides, sugars, lipid moiety) typically are retainedon exposure to denaturing solvents whereas epitopes formed by tertiaryfolding typically are lost on treatment with denaturing solvents. Anepitope may include but is not limited to at least 3, at least 5 or 8-10residues (for example, amino acids or nucleotides). In some examples anepitope is less than 20 residues (for example, amino acids ornucleotides) in length, less than 15 residues or less than 12 residues.Two antibodies may bind the same epitope within an antigen if theyexhibit competitive binding for the antigen. In some embodiments, anepitope can be identified by a certain minimal distance to a CDR residueon the antigen-binding molecule. In some embodiments, an epitope can beidentified by the above distance, and further limited to those residuesinvolved in a bond (for example, a hydrogen bond) between an antibodyresidue and an antigen residue. An epitope can be identified by variousscans as well, for example an alanine or arginine scan can indicate oneor more residues that the antigen-binding molecule can interact with.Unless explicitly denoted, a set of residues as an epitope does notexclude other residues from being part of the epitope for a particularantibody. Rather, the presence of such a set designates a minimal series(or set of species) of epitopes. Thus, in some embodiments, a set ofresidues identified as an epitope designates a minimal epitope ofrelevance for the antigen, rather than an exclusive list of residues foran epitope on an antigen.

A “nonlinear epitope” or “conformational epitope” comprisesnoncontiguous polypeptides, amino acids and/or sugars within theantigenic protein to which an antibody specific to the epitope binds. Insome embodiments, at least one of the residues will be noncontiguouswith the other noted residues of the epitope; however, one or more ofthe residues can also be contiguous with the other residues.

A “linear epitope” comprises contiguous polypeptides, amino acids and/orsugars within the antigenic protein to which an antibody specific to theepitope binds. It is noted that, in some embodiments, not every one ofthe residues within the linear epitope need be directly bound (orinvolved in a bond) with the antibody. In some embodiments, linearepitopes can be from immunizations with a peptide that effectivelyconsisted of the sequence of the linear epitope, or from structuralsections of a protein that are relatively isolated from the remainder ofthe protein (such that the antibody can interact, at least primarily),just with that sequence section.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (forexample, bispecific (such as Bi-specific T-cell engagers) andtrispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

The term antibody includes, but is not limited to, fragments that arecapable of binding to an antigen, such as Fv, single-chain Fv (scFv),Fab, Fab′, di-scFv, sdAb (single domain antibody) and (Fab′)₂ (includinga chemically linked F(ab′)2). Papain digestion of antibodies producestwo identical antigen-binding fragments, called “Fab” fragments, eachwith a single antigen-binding site, and a residual “Fc” fragment, whosename reflects its ability to crystallize readily. Pepsin treatmentyields an F(ab′)2 fragment that has two antigen-combining sites and isstill capable of cross-linking antigen. The term antibody also includes,but is not limited to, chimeric antibodies, humanized antibodies, andantibodies of various species such as mouse, human, cynomolgus monkey,etc. Furthermore, for all antibody constructs provided herein, variantshaving the sequences from other organisms are also contemplated. Thus,if a human version of an antibody is disclosed, one of skill in the artwill appreciate how to transform the human sequence based antibody intoa mouse, rat, cat, dog, horse, etc. sequence. Antibody fragments alsoinclude either orientation of single chain scFvs, tandem di-scFv,diabodies, tandem tri-sdcFv, minibodies, etc. Antibody fragments alsoinclude nanobodies (sdAb, an antibody having a single, monomeric domain,such as a pair of variable domains of heavy chains, without a lightchain). An antibody fragment can be referred to as being a specificspecies in some embodiments (for example, human scFv or a mouse scFv).This denotes the sequences of at least part of the non-CDR regions,rather than the source of the construct.

The term “monoclonal antibody” refers to an antibody of a substantiallyhomogeneous population of antibodies, that is, the individual antibodiescomprising the population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.Monoclonal antibodies are highly specific, being directed against asingle antigenic site. Furthermore, in contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. Thus, a sample ofmonoclonal antibodies can bind to the same epitope on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies may be made bythe hybridoma method first described by Kohler and Milstein, 1975,Nature 256:495, or may be made by recombinant DNA methods such asdescribed in U.S. Pat. No. 4,816,567. The monoclonal antibodies may alsobe isolated from phage libraries generated using the techniquesdescribed in McCafferty et al., 1990, Nature 348:552-554, for example.

The term “CDR” denotes a complementarity determining region as definedby at least one manner of identification to one of skill in the art. Insome embodiments, CDRs can be defined in accordance with any of theChothia numbering schemes, the Kabat numbering scheme, a combination ofKabat and Chothia, the AbM definition, the contact definition, and/or acombination of the Kabat, Chothia, AbM, and/or contact definitions.Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3)occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3,31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The AbM definitioncan include, for example, CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2,and CDR-H3) at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 ofL3, H26-H35B of H1, 50-58 of H2, and 95-102 of H3. The Contactdefinition can include, for example, CDRs (CDR-L1, CDR-L2, CDR-L3,CDR-H1, CDR-H2, and CDR-H3) at amino acid residues 30-36 of L1, 46-55 ofL2, 89-96 of L3, 30-35 of H1, 47-58 of H2, and 93-101 of H3. The Chothiadefinition can include, for example, CDRs (CDR-L1, CDR-L2, CDR-L3,CDR-H1, CDR-H2, and CDR-H3) at amino acid residues 24-34 of L1, 50-56 ofL2, 89-97 of L3, 26-32 . . . 34 of H1, 52-56 of H2, and 95-102 of H3.CDRs can also be provided as shown in any one or more of theaccompanying figures. With the exception of CDR1 in V_(H), CDRsgenerally comprise the amino acid residues that form the hypervariableloops. The various CDRs within an antibody can be designated by theirappropriate number and chain type, including, without limitation as: a)CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3; b) CDRL1, CDRL2,CDRL3, CDRH1, CDRH2, and CDRH3; c) LCDR-1, LCDR-2, LCDR-3, HCDR-1,HCDR-2, and HCDR-3; or d) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3;etc. The term “CDR” is used herein to also encompass HVR or a “hypervariable region”, including hypervariable loops. Exemplary hypervariableloops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3),26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol.Biol. 196:901-917 (1987).)

The term “heavy chain variable region” as used herein refers to a regioncomprising at least three heavy chain CDRs. In some embodiments, theheavy chain variable region includes the three CDRs and at least FR2 andFR3. In some embodiments, the heavy chain variable region includes atleast heavy chain HCDR1, framework (FR) 2, HCDR2, FR3, and HCDR3. Insome embodiments, a heavy chain variable region also comprises at leasta portion of an FR1 and/or at least a portion of an FR4.

The term “heavy chain constant region” as used herein refers to a regioncomprising at least three heavy chain constant domains, C_(H)1, C_(H)2,and C_(H)3. Of course, non-function-altering deletions and alterationswithin the domains are encompassed within the scope of the term “heavychain constant region,” unless designated otherwise. Nonlimitingexemplary heavy chain constant regions include γ, δ, and α. Nonlimitingexemplary heavy chain constant regions also include ε and μ. Each heavyconstant region corresponds to an antibody isotype. For example, anantibody comprising a γ constant region is an IgG antibody, an antibodycomprising a δ constant region is an IgD antibody, and an antibodycomprising an a constant region is an IgA antibody. Further, an antibodycomprising α constant region is an IgM antibody, and an antibodycomprising an ε constant region is an IgE antibody. Certain isotypes canbe further subdivided into subclasses. For example, IgG antibodiesinclude, but are not limited to, IgG1 (comprising a γ₁ constant region),IgG2 (comprising a 72 constant region), IgG3 (comprising a γ₃ constantregion), and IgG4 (comprising a 74 constant region) antibodies; IgAantibodies include, but are not limited to, IgA1 (comprising an α₁constant region) and IgA2 (comprising an α₂ constant region) antibodies;and IgM antibodies include, but are not limited to, IgM1 and IgM2.

The term “heavy chain” as used herein refers to a polypeptide comprisingat least a heavy chain variable region, with or without a leadersequence. In some embodiments, a heavy chain comprises at least aportion of a heavy chain constant region. The term “full-length heavychain” as used herein refers to a polypeptide comprising a heavy chainvariable region and a heavy chain constant region, with or without aleader sequence.

The term “light chain variable region” as used herein refers to a regioncomprising at least three light chain CDRs. In some embodiments, thelight chain variable region includes the three CDRs and at least FR2 andFR3. In some embodiments, the light chain variable region includes atleast light chain LCDR1, framework (FR) 2, LCDR2, FR3, and LCDR3. Forexample, a light chain variable region may comprise light chain CDR1,framework (FR) 2, CDR2, FR3, and CDR3. In some embodiments, a lightchain variable region also comprises at least a portion of an FR1 and/orat least a portion of an FR4.

The term “light chain constant region” as used herein refers to a regioncomprising a light chain constant domain, CL. Nonlimiting exemplarylight chain constant regions include X and K. Of course,non-function-altering deletions and alterations within the domains areencompassed within the scope of the term “light chain constant region,”unless designated otherwise.

The term “light chain” as used herein refers to a polypeptide comprisingat least a light chain variable region, with or without a leadersequence. In some embodiments, a light chain comprises at least aportion of a light chain constant region. The term “full-length lightchain” as used herein refers to a polypeptide comprising a light chainvariable region and a light chain constant region, with or without aleader sequence.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain(V_(L)) framework or a heavy chain variable domain (V_(H)) frameworkderived from a human immunoglobulin framework or a human consensusframework, as defined below. An acceptor human framework derived from ahuman immunoglobulin framework or a human consensus framework cancomprise the same amino acid sequence thereof, or it can contain aminoacid sequence changes. In some embodiments, the number of amino acidchanges are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 orless, 4 or less, 3 or less, or 2 or less. In some embodiments, the V_(L)acceptor human framework is identical in sequence to the V_(L) humanimmunoglobulin framework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (for example,an antibody) and its binding partner (for example, an antigen). Theaffinity of a molecule X for its partner Y can generally be representedby the dissociation constant (K_(D)). Affinity can be measured by commonmethods known in the art (such as, for example, ELISA K_(D), KinExA,bio-layer interferometry (BLI), and/or surface plasmon resonance devices(such as a BIAcore® device), including those described herein).

The term “K_(D)”, as used herein, refers to the equilibrium dissociationconstant of an antibody-antigen interaction.

In some embodiments, the “K_(D),” “Kd,” “Kd” or “Kd value” of theantibody is measured by using surface plasmon resonance assays using aBIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at25° C. with immobilized antigen CM5 chips at ˜10 response units (RU).Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.)are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μL/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, serialdilutions of polypeptide, for example, full length antibody, areinjected in PBS with 0.05% TWEEN-20™ surfactant (PBST) at 25° C. at aflow rate of approximately 25 μL/min. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (K_(d)) is calculated as the ratiok_(off)/k_(on). See, for example, Chen et al., J. Mol. Biol. 293:865-881(1999). If the on-rate exceeds 10⁶ M⁻¹s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody in PBS, pH7.2, in the presence of increasing concentrations of antigen as measuredin a spectrometer, such as a stop-flow equipped spectrophometer (AvivInstruments) or a 8000-series SLM-AMINCO™ spectrophotometer(ThermoSpectronic) with a stirred cuvette.

In some embodiments, the difference between said two values (forexample, K_(d) values) is substantially the same, for example, less thanabout 50%, less than about 40%, less than about 30%, less than about20%, and/or less than about 10% as a function of thereference/comparator value.

In some embodiments, the difference between said two values (forexample, K_(d) values) is substantially different, for example, greaterthan about 10%, greater than about 20%, greater than about 30%, greaterthan about 40%, and/or greater than about 50% as a function of the valuefor the reference/comparator molecule.

“Surface plasmon resonance” denotes an optical phenomenon that allowsfor the analysis of real-time biospecific interactions by detection ofalterations in protein concentrations within a biosensor matrix, forexample using the BIAcore™ system (BIAcore International AB, a GEHealthcare company, Uppsala, Sweden and Piscataway, N.J.). For furtherdescriptions, see Jonsson et al. (1993) Ann. Biol. Clin. 51:19-26.

“Biolayer interferometry” refers to an optical analytical technique thatanalyzes the interference pattern of light reflected from a layer ofimmobilized protein on a biosensor tip and an internal reference layer.Changes in the number of molecules bound to the biosensor tip causeshifts in the interference pattern that can be measured in real-time. Anonlimiting exemplary device for biolayer interferometry is ForteBioOctet® RED96 system (Pall Corporation). See, e.g., Abdiche et al., 2008,Anal. Biochem. 377: 209-277.

The term “k_(on)”, as used herein, refers to the rate constant forassociation of an antibody to an antigen. Specifically, the rateconstants (k_(on) and k_(off)) and equilibrium dissociation constantsare measured using IgGs (bivalent) with monovalent PD-1 antigen.“K_(on)”, “k_(on)”, “association rate constant”, or “ka”, are usedinterchangeably herein. The value indicates the binding rate of abinding protein to its target antigen or the rate of complex formationbetween an antibody and antigen, shown by the equation:Antibody(“Ab”)+Antigen(“Ag”)→Ab−Ag. The term “k_(off)”, as used herein,refers to the rate constant for dissociation of an antibody from theantibody/antigen complex. k_(off) is also denoted as “K_(off)” or the“dissociation rate constant”. This value indicates the dissociation rateof an antibody from its target antigen or separation of Ab−Ag complexover time into free antibody and antigen as shown by the equation:

Ab+Ag←Ab−Ag.

The term “biological activity” refers to any one or more biologicalproperties of a molecule (whether present naturally as found in vivo, orprovided or enabled by recombinant means). Biological propertiesinclude, but are not limited to, binding a receptor, inducing cellproliferation, inhibiting cell growth, inducing other cytokines,inducing apoptosis, and enzymatic activity. In some embodiments,biological activity of a PD-1 protein includes, for example, promotingapoptosis in antigen-specific T cells, reducing apoptosis in regulatoryT (Treg) cells, inhibiting activation of T cells, inhibitingproliferation of T cells, and facilitating T cell anergy or exhaustion.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more CDRs compared to a parent antibody which doesnot possess such alterations, such alterations resulting in animprovement in the affinity of the antibody for antigen.

A “chimeric antibody” as used herein refers to an antibody in which aportion of the heavy and/or light chain is derived from a particularsource or species, while at least a part of the remainder of the heavyand/or light chain is derived from a different source or species. Insome embodiments, a chimeric antibody refers to an antibody comprisingat least one variable region from a first species (such as mouse, rat,cynomolgus monkey, etc.) and at least one constant region from a secondspecies (such as human, cynomolgus monkey, etc.). In some embodiments, achimeric antibody comprises at least one mouse variable region and atleast one human constant region. In some embodiments, a chimericantibody comprises at least one cynomolgus variable region and at leastone human constant region. In some embodiments, all of the variableregions of a chimeric antibody are from a first species and all of theconstant regions of the chimeric antibody are from a second species. Thechimeric construct can also be a functional fragment, as noted above.

A “humanized antibody” as used herein refers to an antibody in which atleast one amino acid in a framework region of a non-human variableregion has been replaced with the corresponding amino acid from a humanvariable region. In some embodiments, a humanized antibody comprises atleast one human constant region or fragment thereof. In someembodiments, a humanized antibody is an antibody fragment, such as Fab,an scFv, a (Fab′)₂, etc. The term humanized also denotes forms ofnon-human (for example, murine) antibodies that are chimericimmunoglobulins, immunoglobulin chains, or fragments thereof (such asFv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences ofantibodies) that contain minimal sequence of non-human immunoglobulin.Humanized antibodies can include human immunoglobulins (recipientantibody) in which residues from a complementary determining region(CDR) of the recipient are substituted by residues from a CDR of anon-human species (donor antibody) such as mouse, rat, or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, the humanized antibodycan comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences, but are included tofurther refine and optimize antibody performance. In general, thehumanized antibody can comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. In some embodiments, the humanizedantibody can also comprise at least a portion of an immunoglobulinconstant region or domain (Fc), typically that of a humanimmunoglobulin. Other forms of humanized antibodies have one or moreCDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and/or CDR H3) which arealtered with respect to the original antibody, which are also termed oneor more CDRs “derived from” one or more CDRs from the original antibody.As will be appreciated, a humanized sequence can be identified by itsprimary sequence and does not necessarily denote the process by whichthe antibody was created.

An “CDR-grafted antibody” as used herein refers to a humanized antibodyin which one or more complementarity determining regions (CDRs) of afirst (non-human) species have been grafted onto the framework regions(FRs) of a second (human) species.

A “human antibody” as used herein encompasses antibodies produced inhumans, antibodies produced in non-human animals that comprise humanimmunoglobulin genes, such as XenoMouse® mice, and antibodies selectedusing in vitro methods, such as phage display (Vaughan et al., 1996,Nature Biotechnology, 14:309-314; Sheets et al., 1998, Proc. Natl. Acad.Sci. (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol.,227:381; Marks et al., 1991, J. Mol. Biol., 222:581), wherein theantibody repertoire is based on a human immunoglobulin sequence. Theterm “human antibody” denotes the genus of sequences that are humansequences. Thus, the term is not designating the process by which theantibody was created, but the genus of sequences that are relevant.

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include Fc receptorbinding; Clq binding; CDC; ADCC; phagocytosis; down regulation of cellsurface receptors (for example B cell receptor; BCR), etc. Such effectorfunctions generally require the Fc region to be combined with a bindingdomain (for example, an antibody variable domain) and can be assessedusing various assays.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification. In some embodiments, a “variant Fc region” comprisesan amino acid sequence which differs from that of a native sequence Fcregion by virtue of at least one amino acid modification, yet retains atleast one effector function of the native sequence Fc region. In someembodiments, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, for example, from about one to about ten aminoacid substitutions, and preferably, from about one to about five aminoacid substitutions in a native sequence Fc region or in the Fc region ofthe parent polypeptide. In some embodiments, the variant Fc regionherein will possess at least about 80% sequence identity with a nativesequence Fc region and/or with an Fc region of a parent polypeptide, atleast about 90% sequence identity therewith, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity therewith.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. In some embodiments, an FcγR is a native human FcR. Insome embodiments, an FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof those receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domainInhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see, for example,Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, forexample, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capelet al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.Med. 126:330-41 (1995). Other FcRs, including those to be identified inthe future, are encompassed by the term “FcR” herein.

The term “Fe receptor” or “FcR” also includes the neonatal receptor,FcRn, which is responsible for the transfer of maternal IgGs to thefetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)) and regulation of homeostasis ofimmunoglobulins. Methods of measuring binding to FcRn are known (see,for example, Ghetie and Ward., Immunol. Today 18(12):592-598 (1997);Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton etal., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004/92219 (Hinton etal.).

“Effector functions” refer to biological activities attributable to theFc region of an antibody, which vary with the antibody isotype. Examplesof antibody effector functions include: Clq binding and complementdependent cytotoxicity (CDC); Fc receptor binding; antibody-dependentcell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cellsurface receptors (for example B cell receptor); and B cell activation.

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. In some embodiments, the cells express atleast FcγRIII and perform ADCC effector function(s). Examples of humanleukocytes which mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, andneutrophils. The effector cells may be isolated from a native source,for example, from blood.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (for example NK cells, neutrophils,and macrophages) enable these cytotoxic effector cells to bindspecifically to an antigen-bearing target cell and subsequently kill thetarget cell with cytotoxins. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII,and FcγRIII. FcR expression on hematopoietic cells is summarized inTable 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92(1991). To assess ADCC activity of a molecule of interest, an in vitroADCC assay, such as that described in U.S. Pat. No. 5,500,362 or5,821,337 or 6,737,056 (Presta), may be performed. Useful effector cellsfor such assays include PBMC and NK cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, for example, in an animal model such as that disclosed inClynes et al. Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998). Additionalpolypeptide variants with altered Fc region amino acid sequences(polypeptides with a variant Fc region) and increased or decreased ADCCactivity are described, for example, in U.S. Pat. Nos. 7,923,538, and7,994,290.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (Clq) to antibodies (of the appropriate subclass),which are bound to their cognate antigen. To assess complementactivation, a CDC assay, for example, as described in Gazzano-Santoro etal., J. Immunol. Methods 202:163 (1996), may be performed. Polypeptidevariants with altered Fc region amino acid sequences (polypeptides witha variant Fc region) and increased or decreased Clq binding capabilityare described, for example, in U.S. Pat. No. 6,194,551 B1, U.S. Pat.Nos. 7,923,538, 7,994,290 and WO 1999/51642. See also, for example,Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

A polypeptide variant with “altered” FcR binding affinity or ADCCactivity is one which has either enhanced or diminished FcR bindingactivity and/or ADCC activity compared to a parent polypeptide or to apolypeptide comprising a native sequence Fc region. The polypeptidevariant which “displays increased binding” to an FcR binds at least oneFcR with better affinity than the parent polypeptide. The polypeptidevariant which “displays decreased binding” to an FcR, binds at least oneFcR with lower affinity than a parent polypeptide. Such variants whichdisplay decreased binding to an FcR may possess little or no appreciablebinding to an FcR, for example, 0-20% binding to the FcR compared to anative sequence IgG Fc region.

The polypeptide variant which “mediates antibody-dependent cell-mediatedcytotoxicity (ADCC) in the presence of human effector cells moreeffectively” than a parent antibody is one which in vitro or in vivo ismore effective at mediating ADCC, when the amounts of polypeptidevariant and parent antibody used in the assay are essentially the same.Generally, such variants will be identified using the in vitro ADCCassay as herein disclosed, but other assays or methods for determiningADCC activity, for example in an animal model etc., are contemplated.

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between two ormore numeric values such that one of skill in the art would consider thedifference between the two or more values to be of little or nobiological and/or statistical significance within the context of thebiological characteristic measured by said value. In some embodimentsthe two or more substantially similar values differ by no more thanabout any one of 5%, 10%, 15%, 20%, 25%, or 50%.

The phrase “substantially different,” as used herein, denotes asufficiently high degree of difference between two numeric values suchthat one of skill in the art would consider the difference between thetwo values to be of statistical significance within the context of thebiological characteristic measured by said values. In some embodiments,the two substantially different numeric values differ by greater thanabout any one of 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%,90%, or 100%.

The phrase “substantially reduced,” as used herein, denotes asufficiently high degree of reduction between a numeric value and areference numeric value such that one of skill in the art would considerthe difference between the two values to be of statistical significancewithin the context of the biological characteristic measured by saidvalues. In some embodiments, the substantially reduced numeric values isreduced by greater than about any one of 10%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the reference value.

The term “leader sequence” refers to a sequence of amino acid residueslocated at the N-terminus of a polypeptide that facilitates secretion ofa polypeptide from a mammalian cell. A leader sequence can be cleavedupon export of the polypeptide from the mammalian cell, forming a matureprotein. Leader sequences can be natural or synthetic, and they can beheterologous or homologous to the protein to which they are attached.

A “native sequence” polypeptide comprises a polypeptide having the sameamino acid sequence as a polypeptide found in nature. Thus, a nativesequence polypeptide can have the amino acid sequence of naturallyoccurring polypeptide from any mammal. Such native sequence polypeptidecan be isolated from nature or can be produced by recombinant orsynthetic means. The term “native sequence” polypeptide specificallyencompasses naturally occurring truncated or secreted forms of thepolypeptide (for example, an extracellular domain sequence), naturallyoccurring variant forms (for example, alternatively spliced forms) andnaturally occurring allelic variants of the polypeptide.

A polypeptide “variant” means a biologically active polypeptide havingat least about 80% amino acid sequence identity with the native sequencepolypeptide after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Such variants include, for instance, polypeptides wherein oneor more amino acid residues are added, or deleted, at the N- orC-terminus of the polypeptide. In some embodiments, a variant will haveat least about 80% amino acid sequence identity. In some embodiments, avariant will have at least about 90% amino acid sequence identity. Insome embodiments, a variant will have at least about 95% amino acidsequence identity with the native sequence polypeptide.

As used herein, “Percent (%) amino acid sequence identity” and“homology” with respect to a peptide, polypeptide or antibody sequenceare defined as the percentage of amino acid residues in a candidatesequence that are identical with the amino acid residues in the specificpeptide or polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.

An amino acid substitution may include but are not limited to thereplacement of one amino acid in a polypeptide with another amino acid.Exemplary substitutions are shown in Table 1. Amino acid substitutionsmay be introduced into an antibody of interest and the products screenedfor a desired activity, for example, retained/improved antigen binding,decreased immunogenicity, or improved ADCC or CDC.

TABLE 1 Original Residue Exemplary Substitutions Ala (A) Val; Leu; IleArg (R) Lys; Gln; Asn Asn (N) Gln; His; Asp, Lys; Arg Asp (D) Glu; AsnCys (C) Ser; Ala Gln (Q) Asn; Glu Glu (E) Asp; Gln Gly (G) Ala His (H)Asn; Gln; Lys; Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu (L)Norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg; Gln; Asn Met (M) Leu;Phe; Ile Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ser (S) ThrThr (T) Val; Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe; Thr; Ser Val (V)Ile; Leu; Met; Phe; Ala; Norleucine

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

The term “vector” is used to describe a polynucleotide that can beengineered to contain a cloned polynucleotide or polynucleotides thatcan be propagated in a host cell. A vector can include one or more ofthe following elements: an origin of replication, one or more regulatorysequences (such as, for example, promoters and/or enhancers) thatregulate the expression of the polypeptide of interest, and/or one ormore selectable marker genes (such as, for example, antibioticresistance genes and genes that can be used in colorimetric assays, forexample, β-galactosidase). The term “expression vector” refers to avector that is used to express a polypeptide of interest in a host cell.

A “host cell” refers to a cell that may be or has been a recipient of avector or isolated polynucleotide. Host cells may be prokaryotic cellsor eukaryotic cells. Exemplary eukaryotic cells include mammalian cells,such as primate or non-primate animal cells; fungal cells, such asyeast; plant cells; and insect cells. Nonlimiting exemplary mammaliancells include, but are not limited to, NSO cells, PER.C6® cells(Crucell), and 293 and CHO cells, and their derivatives, such as 293-6Eand DG44 cells, respectively. Host cells include progeny of a singlehost cell, and the progeny may not necessarily be completely identical(in morphology or in genomic DNA complement) to the original parent celldue to natural, accidental, or deliberate mutation. A host cell includescells transfected in vivo with a polynucleotide(s) a provided herein.

The term “isolated” as used herein refers to a molecule that has beenseparated from at least some of the components with which it istypically found in nature or produced. For example, a polypeptide isreferred to as “isolated” when it is separated from at least some of thecomponents of the cell in which it was produced. Where a polypeptide issecreted by a cell after expression, physically separating thesupernatant containing the polypeptide from the cell that produced it isconsidered to be “isolating” the polypeptide. Similarly, apolynucleotide is referred to as “isolated” when it is not part of thelarger polynucleotide (such as, for example, genomic DNA ormitochondrial DNA, in the case of a DNA polynucleotide) in which it istypically found in nature, or is separated from at least some of thecomponents of the cell in which it was produced, for example, in thecase of an RNA polynucleotide. Thus, a DNA polynucleotide that iscontained in a vector inside a host cell may be referred to as“isolated”.

The terms “individual” or “subject” are used interchangeably herein torefer to an animal; for example, a mammal. In some embodiments, methodsof treating mammals, including, but not limited to, humans, rodents,simians, felines, canines, equines, bovines, porcines, ovines, caprines,mammalian laboratory animals, mammalian farm animals, mammalian sportanimals, and mammalian pets, are provided. In some examples, an“individual” or “subject” refers to an individual or subject in need oftreatment for a disease or disorder. In some embodiments, the subject toreceive the treatment can be a patient, designating the fact that thesubject has been identified as having a disorder of relevance to thetreatment, or being at adequate risk of contracting the disorder.

A “disease” or “disorder” as used herein refers to a condition wheretreatment is needed and/or desired.

“Cancer” and “tumor,” as used herein, are interchangeable terms thatrefer to any abnormal cell or tissue growth or proliferation in ananimal. As used herein, the terms “cancer” and “tumor” encompass solidand hematological/lymphatic cancers and also encompass malignant,pre-malignant, and benign growth, such as dysplasia. Examples of cancerinclude but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia. More particular non-limiting examples of such cancersinclude squamous cell cancer, small-cell lung cancer, pituitary cancer,esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lungcancer, adenocarcinoma of the lung, squamous carcinoma of the lung,cancer of the peritoneum, hepatocellular cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, colorectal cancer, endometrial or uterine carcinoma, salivarygland carcinoma, kidney cancer, renal cancer, liver cancer, prostatecancer, vulval cancer, thyroid cancer, hepatic carcinoma, brain cancer,endometrial cancer, testis cancer, cholangiocarcinoma, gallbladdercarcinoma, gastric cancer, melanoma, mesothelioma, and various types ofhead and neck cancer.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. “Treatment” as used herein, covers anyadministration or application of a therapeutic for disease in a mammal,including a human. For purposes of this disclosure, beneficial ordesired clinical results include, but are not limited to, any one ormore of: alleviation of one or more symptoms, diminishment of extent ofdisease, preventing or delaying spread (for example, metastasis, forexample metastasis to the lung or to the lymph node) of disease,preventing or delaying recurrence of disease, delay or slowing ofdisease progression, amelioration of the disease state, inhibiting thedisease or progression of the disease, inhibiting or slowing the diseaseor its progression, arresting its development, and remission (whetherpartial or total). Also encompassed by “treatment” is a reduction ofpathological consequence of a proliferative disease. The methodsprovided herein contemplate any one or more of these aspects oftreatment. In-line with the above, the term treatment does not requireone-hundred percent removal of all aspects of the disorder.

“Ameliorating” means a lessening or improvement of one or more symptomsas compared to not administering an anti-PD-1 antibody. “Ameliorating”also includes shortening or reduction in duration of a symptom.

In the context of cancer, the term “treating” includes any or all of:inhibiting growth of cancer cells, inhibiting replication of cancercells, lessening of overall tumor burden and ameliorating one or moresymptoms associated with the disease.

The term “biological sample” means a quantity of a substance from aliving thing or formerly living thing. Such substances include, but arenot limited to, blood, (for example, whole blood), plasma, serum, urine,amniotic fluid, synovial fluid, endothelial cells, leukocytes,monocytes, other cells, organs, tissues, bone marrow, lymph nodes andspleen.

A sample that has an “elevated level of PD-1” or “expresses PD-1 at anelevated level” or is “PD-1^(HIGH)” means that the level of PD-1 is suchthat one of skill in the art would conclude that the cancer may betreatable with an anti-PD-1 therapy, such as an antibody providedherein. In some embodiments, an “elevated level of PD-1” is one in which1% of the cells within a tumor sample show staining for PD-1. In someembodiments a “high level” in regard to PD-1 is 1% or more staining, forexample, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the cellswithin the tumor sample show staining. In some embodiments, the PD-1levels can be measured by chromogenic IHC or immunofluorescence IHC(Aqua scoring).

A sample that “expresses PD-1” or has “positive staining for PD-1” or is“PD-1 positive” means that 1% or more of the cells in a sample showstaining for PD-1. In some embodiments, a sample that is PD-1 positivedisplays at least weak, moderate, and/or strong cell staining (based onmembrane expression of PD-1). A sample with moderate or strong cellstaining for PD-1 is also considered to be “PD-1^(HIGH).”

A sample that has a “low level of PD-L1” or expresses “PD-L1 at a lowlevel” or is “PD-L1^(LOW)” means that the level of PD-L1 is below thethreshold level of expression for a cancer that is normally indicatedfor treatment with a PD-1 therapy. In some embodiments, a “low level ofPD-L1” is one in which less than 5% of the cells in the tumor showmembrane staining for PD-L1. In some embodiments a “low level” in regardto PD-L1 is less than 5% staining, for example, 4%, 3%, 2%, 1%, or 0% ofthe cells of the tumor show staining. In some embodiments, the PD-L1levels can be measured by chromogenic IHC or immunofluorescence IHC(Aqua scoring). A sample that expresses no detectable PD-L1 can also besaid to “express a low level of PD-L1.” Thus, no detectable PD-L1 isencompassed within the term “low.”

A sample that has an “elevated level of PD-L1” or “expresses PD-L1 at anelevated level” or is “PD-L1^(HIGH)” means that the level of PD-L1 thatis such that one of skill in the art would conclude that the cancer maybe treatable with a PD-1 therapy. In some embodiments, an “elevatedlevel of PD-L1” is one in which 50% of the cells in the tumor or morehave membrane staining of PD-L1, for example, 50, 60, 70, 80, 90, or100% of the cells of the tumor show membrane staining. In someembodiments, the PD-L1 levels can be measured by chromogenic IHC orimmunofluorescence IHC (AQUA scoring).

A sample that “expresses PD-L1” or has “positive staining for PD-L1” oris “PD-L1 positive” means that 1% or more of the cells have membranestaining for PD-L1. In some embodiments, a sample that is PD-L1 positivedisplays at least weak, moderate, and/or strong cell staining (based onmembrane expression of PD-L1).

A sample that “lacks PD-L1 expression” or has “negative staining forPD-L1” or is “PD-L1 negative” means that PD-L1 expression on the surfaceof cells of the sample is undetectable by IHC, such as chromogenic IHCor immunofluorescence IHC (Aqua scoring). A PD-L1 negative sample isalso be considered to be “PD-L1^(LOW).”

In some embodiments, any method for measuring the level of PD-L1 can beemployed. In some embodiments, this can include using the PD-L1 IHC 22C3pharmDx test (Dako, Inc., Carpinteria, Calif.), which is a clinicallyvalidated and FDA approved test for evaluation of PD-L1 expression inNSCLC. PD-L1 IHC 22C3 pharmDx is a qualitative immunohistochemical assayusing monoclonal mouse anti-PD-L1 antibody, (clone 22C3), that can beused in the detection of PD-L1 protein in formalin-fixedparaffin-embedded (FFPE) Non-Small Cell Lung Cancer (NSCLC) tissues. Theassay can be performed on Autostainer Link 48 system and visualizedusing the EnVision FLEX system. PD-L1 protein expression is qualifiedusing Tumor Proportion Score (TPS), which is the percentage of viabletumor cells showing partial or complete membrane staining. In someembodiments, the specimen is considered PD-L1 positive if TPS≥1% of theviable tumor cells exhibit membrane staining at any intensity. In someembodiments, the specimen is considered PD-L1^(HIGH) if TPS≥50% of theviable tumor cells exhibit membrane staining at any intensity. PD-L1 IHC22C3 pharmDx is indicated as an aid in identifying NSCLC patients fortreatment with KEYTRUDA® (pembrolizumab). Additional details on thescoring system and response to pembrolizumab are described in thearticle by Garon et al. (N Engl J Med 2015; 372:2018-28).

The term “control” refers to a composition known to not contain ananalyte (“negative control”) or to contain analyte (“positive control”).A positive control can comprise a known concentration of analyte.“Control,” “positive control,” and “calibrator” may be usedinterchangeably herein to refer to a composition comprising a knownconcentration of analyte. A “positive control” can be used to establishassay performance characteristics and is a useful indicator of theintegrity of reagents (for example, analytes).

“Predetermined cutoff” and “predetermined level” refer generally to anassay cutoff value that is used to assessdiagnostic/prognostic/therapeutic efficacy results by comparing theassay results against the predetermined cutoff/level, where thepredetermined cutoff/level already has been linked or associated withvarious clinical parameters (for example, severity of disease,progression/nonprogression/improvement, etc.). While the presentdisclosure may provide exemplary predetermined levels, it is well-knownthat cutoff values may vary depending on the nature of the immunoassay(for example, antibodies employed, etc.). It further is well within theskill of one of ordinary skill in the art to adapt the disclosure hereinfor other immunoassays to obtain immunoassay-specific cutoff values forthose other immunoassays based on this disclosure. Whereas the precisevalue of the predetermined cutoff/level may vary between assays,correlations as described herein (if any) may be generally applicable.

The terms “inhibition” or “inhibit” refer to a decrease or cessation ofany phenotypic characteristic or to the decrease or cessation in theincidence, degree, or likelihood of that characteristic. To “reduce” or“inhibit” is to decrease, reduce or arrest an activity, function, and/oramount as compared to a reference. In some embodiments, by “reduce” or“inhibit” is meant the ability to cause an overall decrease of 20% orgreater. In some embodiments, by “reduce” or “inhibit” is meant theability to cause an overall decrease of 50% or greater. In someembodiments, by “reduce” or “inhibit” is meant the ability to cause anoverall decrease of 75%, 85%, 90%, 95%, or greater. In some embodiments,the amount noted above is inhibited or decreased over a period of time,relative to a control dose (such as a placebo) over the same period oftime. A “reference” as used herein, refers to any sample, standard, orlevel that is used for comparison purposes. A reference may be obtainedfrom a healthy and/or non-diseased sample. In some examples, a referencemay be obtained from an untreated sample. In some examples, a referenceis obtained from a non-diseased on non-treated sample of a subjectindividual. In some examples, a reference is obtained from one or morehealthy individuals who are not the subject or patient.

As used herein, “delaying development of a disease” means to defer,hinder, slow, retard, stabilize, suppress and/or postpone development ofthe disease (such as cancer). This delay can be of varying lengths oftime, depending on the history of the disease and/or individual beingtreated. As is evident to one skilled in the art, a sufficient orsignificant delay can, in effect, encompass prevention, in that theindividual does not develop the disease. For example, a late stagecancer, such as development of metastasis, may be delayed.

“Preventing,” as used herein, includes providing prophylaxis withrespect to the occurrence or recurrence of a disease in a subject thatmay be predisposed to the disease but has not yet been diagnosed withthe disease. Unless otherwise specified, the terms “reduce”, “inhibit”,or “prevent” do not denote or require complete prevention over all time.

As used herein, to “suppress” a function or activity is to reduce thefunction or activity when compared to otherwise same conditions exceptfor a condition or parameter of interest, or alternatively, as comparedto another condition. For example, an antibody which suppresses tumorgrowth reduces the rate of growth of the tumor compared to the rate ofgrowth of the tumor in the absence of the antibody.

A “therapeutically effective amount” of a substance/molecule, agonist orantagonist may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of thesubstance/molecule, agonist or antagonist to elicit a desired responsein the individual. A therapeutically effective amount is also one inwhich any toxic or detrimental effects of the substance/molecule,agonist or antagonist are outweighed by the therapeutically beneficialeffects. A therapeutically effective amount may be delivered in one ormore administrations. A therapeutically effective amount refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired therapeutic and/or prophylactic result.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, but not necessarily, since aprophylactic dose is used in subjects prior to or at an earlier stage ofdisease, the prophylactically effective amount will be less than thetherapeutically effective amount.

The terms “pharmaceutical formulation” and “pharmaceutical composition”refer to a preparation which is in such form as to permit the biologicalactivity of the active ingredient(s) to be effective, and which containsno additional components which are unacceptably toxic to a subject towhich the formulation would be administered. Such formulations may besterile.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid, or liquid filler, diluent, encapsulating material,formulation auxiliary, or carrier conventional in the art for use with atherapeutic agent that together comprise a “pharmaceutical composition”for administration to a subject. A pharmaceutically acceptable carrieris non-toxic to recipients at the dosages and concentrations employedand is compatible with other ingredients of the formulation. Thepharmaceutically acceptable carrier is appropriate for the formulationemployed.

A “sterile” formulation is aseptic or essentially free from livingmicroorganisms and their spores.

The term “IDO inhibitor” refers to an agent capable of inhibiting theactivity of indoleamine 2,3-dioxygenase (IDO) and thereby reversingIDO-mediated immunosuppression. The IDO inhibitor may inhibit IDO1and/or IDO2 (INDOL1). An IDO inhibitor may be a reversible orirreversible IDO inhibitor. A “reversible IDO inhibitor” is a compoundthat reversibly inhibits IDO enzyme activity either at the catalyticsite or at a non-catalytic site and an “irreversible IDO inhibitor” is acompound that irreversibly inhibits IDO enzyme activity by forming acovalent bond with the enzyme. Nonlimiting exemplary IDO inhibitorsinclude Indoximod (New Link Genetics), INCB024360 (Incyte Corp.),1-methyl-D-tryptophan (New Link Genetics), and GDC-0919 (Genentech,Inc.).

A “chimeric antigen receptor T cell therapy” or “CAR-T therapy” refersto a therapeutic agent comprising a T cell genetically modified toexpress a receptor that recognizes an antigen expressed by tumor cell.The antigen may be an antigen specifically expressed by the tumor or anantigen expressed by both cancerous cells and healthy tissue. In someembodiments CAR-T therapy is adoptive CAR-T therapy, in which a patientsT cells are removed and modified to express the chimeric antigenreceptor, and then returned to the patient. See, e.g., Dai et al., 2016,J Natl Cancer Inst, 108 (7): djv439, doi: 10.1093/jnci/djv439; Gill etal., 2015, Blood Rev, pii: S0268-960X(15)00080-6, doi:10.1016/j.blre.2015.10.003; Gill et al., 2015, Immunol Rev,263(1):68-89. doi: 10.1111/imr.12243.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive or sequentialadministration in any order.

The term “concurrently” is used herein to refer to administration of twoor more therapeutic agents, where at least part of the administrationoverlaps in time or where the administration of one therapeutic agentfalls within a short period of time relative to administration of theother therapeutic agent. For example, the two or more therapeutic agentsare administered with a time separation of no more than about aspecified number of minutes.

The term “sequentially” is used herein to refer to administration of twoor more therapeutic agents where the administration of one or moreagent(s) continues after discontinuing the administration of one or moreother agent(s), or wherein administration of one or more agent(s) beginsbefore the administration of one or more other agent(s). For example,administration of the two or more therapeutic agents are administeredwith a time separation of more than about a specified number of minutes.

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during or after administration of the other treatment modalityto the individual.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

An “article of manufacture” is any manufacture (for example, a packageor container) or kit comprising at least one reagent, for example, amedicament for treatment of a disease or disorder (for example, cancer),or a probe for specifically detecting a biomarker described herein. Insome embodiments, the manufacture or kit is promoted, distributed, orsold as a unit for performing the methods described herein.

The terms “label” and “detectable label” mean a moiety attached to anantibody or its analyte to render a reaction (for example, binding)between the members of the specific binding pair, detectable. Thelabeled member of the specific binding pair is referred to as“detectably labeled.” Thus, the term “labeled binding protein” refers toa protein with a label incorporated that provides for the identificationof the binding protein. In some embodiments, the label is a detectablemarker that can produce a signal that is detectable by visual orinstrumental means, for example, incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (for example, streptavidin containing afluorescent marker or enzymatic activity that can be detected by opticalor colorimetric methods). Examples of labels for polypeptides include,but are not limited to, the following: radioisotopes or radionuclides(for example, ³H, ¹⁴C, ³⁵S, ⁹⁰Y ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho,or ¹⁵³Sm); chromogens, fluorescent labels (for example, FITC, rhodamine,lanthanide phosphors), enzymatic labels (for example, horseradishperoxidase, luciferase, alkaline phosphatase); chemiluminescent markers;biotinyl groups; predetermined polypeptide epitopes recognized by asecondary reporter (for example, leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags);and magnetic agents, such as gadolinium chelates. Representativeexamples of labels commonly employed for immunoassays include moietiesthat produce light, for example, acridinium compounds, and moieties thatproduce fluorescence, for example, fluorescein. In this regard, themoiety itself may not be detectably labeled but may become detectableupon reaction with yet another moiety.

The term “conjugate” refers to an antibody that is chemically linked toa second chemical moiety, such as a therapeutic or cytotoxic agent. Theterm “agent” includes a chemical compound, a mixture of chemicalcompounds, a biological macromolecule, or an extract made frombiological materials. In some embodiments, the therapeutic or cytotoxicagents include, but are not limited to, pertussis toxin, taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. When employed in the context of an immunoassay, theconjugate antibody may be a detectably labeled antibody used as thedetection antibody.

II. Anti-PD-1 Antibodies

Novel antibodies directed against PD-1 are provided. Anti-PD-1antibodies include, but are not limited to, humanized antibodies,chimeric antibodies, mouse antibodies, human antibodies, and antibodiescomprising the heavy chain and/or light chain CDRs discussed herein. Insome embodiments, an isolated antibody that binds to PD-1 is provided.In some embodiments, a monoclonal antibody that binds to PD-1 isprovided. In some embodiments, an anti-PD-1 antibody is an antagonistanti-PD-1 antibody. In some embodiments, an anti-PD-1 antibody providedherein inhibits binding of PD-1 to PD-L1 and/or PD-L2. In someembodiments, an anti-PD-1 antibody provided herein inhibits binding ofPD-1 to PD-L1. In some embodiments, an anti-PD-1 antibody providedherein inhibits binding of PD-1 to PD-L1 and PD-L2. In some embodiments,administration of the anti-PD-1 antibodies described herein enhances animmune response in a subject, and/or increases activation of T cells ina subject.

In some embodiments, an anti-PD-1 antibody comprises at least one, two,three, four, five, or six CDRs selected from (a) HCDR1 comprising theamino acid sequence of SEQ ID NO: 5; (b) HCDR2 comprising the amino acidsequence of SEQ ID NO: 6; (c) HCDR3 comprising the amino acid sequenceof SEQ ID NO: 7; (d) LCDR1 comprising the amino acid sequence of SEQ IDNO: 9; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO: 10;and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO: 11.

In some embodiments, an anti-PD-1 antibody comprises at least one, two,three, four, five, or six CDRs selected from (a) HCDR1 comprising theamino acid sequence of SEQ ID NO: 13; (b) HCDR2 comprising the aminoacid sequence of SEQ ID NO: 14; (c) HCDR3 comprising the amino acidsequence of SEQ ID NO: 15; (d) LCDR1 comprising the amino acid sequenceof SEQ ID NO: 17; (e) LCDR2 comprising the amino acid sequence of SEQ IDNO: 18; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO:19.

In some embodiments, an anti-PD-1 antibody comprises at least one, two,three, four, five, or six CDRs selected from (a) HCDR1 comprising theamino acid sequence of SEQ ID NO: 21; (b) HCDR2 comprising the aminoacid sequence of SEQ ID NO: 22; (c) HCDR3 comprising the amino acidsequence of SEQ ID NO: 23; (d) LCDR1 comprising the amino acid sequenceof SEQ ID NO: 25; (e) LCDR2 comprising the amino acid sequence of SEQ IDNO: 26; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO:27.

In some embodiments, an anti-PD-1 antibody comprises at least one, two,three, four, five, or six CDRs selected from (a) HCDR1 comprising theamino acid sequence of SEQ ID NO: 29; (b) HCDR2 comprising the aminoacid sequence of SEQ ID NO: 30; (c) HCDR3 comprising the amino acidsequence of SEQ ID NO: 31; (d) LCDR1 comprising the amino acid sequenceof SEQ ID NO: 33; (e) LCDR2 comprising the amino acid sequence of SEQ IDNO: 34; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO:35.

In some embodiments, an anti-PD-1 antibody comprises at least one, two,three, four, five, or six CDRs selected from (a) HCDR1 comprising theamino acid sequence of SEQ ID NO: 37; (b) HCDR2 comprising the aminoacid sequence of SEQ ID NO: 38; (c) HCDR3 comprising the amino acidsequence of SEQ ID NO: 39; (d) LCDR1 comprising the amino acid sequenceof SEQ ID NO: 41; (e) LCDR2 comprising the amino acid sequence of SEQ IDNO: 42; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO:43.

In some embodiments, an anti-PD-1 antibody comprises at least one, two,three, four, five, or six CDRs selected from (a) HCDR1 comprising anamino acid sequence selected from SEQ ID NOs: 45, 53, 61, 69, 77, 85,93, 101, 109 and 117; (b) HCDR2 comprising an amino acid sequenceselected from SEQ ID NOs: 46, 54, 62, 70, 78, 86, 94, 102, 110 and 118;(c) HCDR3 comprising an amino acid sequence selected from SEQ ID NOs:47, 55, 63, 71, 79, 87, 95, 103, 111, and 119; (d) LCDR1 comprising anamino acid sequence selected from SEQ ID NOs: 49, 57, 65, 73, 81, 89,97, 105, 113, and 121; (e) LCDR2 comprising an amino acid sequenceselected from SEQ ID NOs: 50, 58, 66, 74, 82, 90, 98, 106, 114, and 122;and (f) LCDR3 comprising an amino acid sequence selected from SEQ IDNOs: 51, 59, 67, 75, 83, 91, 99, 107, 115, and 123.

In some embodiments, an anti-PD-1 antibody comprises at least one, two,three, four, five, or six CDRs selected from (a) HCDR1 comprising anamino acid sequence selected from SEQ ID NOs: 125, 133, 141, 149, 157,165, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, 269,277 and 285; (b) HCDR2 comprising an amino acid sequence selected fromSEQ ID NOs: 126, 134, 142, 150, 158, 166, 174, 182, 190, 198, 206, 214,222, 230, 238, 246, 254, 262, 270, 278 and 286; (c) HCDR3 comprising anamino acid sequence selected from SEQ ID NOs: 127, 135, 143, 151, 159,167, 175, 183, 191, 199, 207, 215, 223, 231, 239, 247, 255, 263, 271,279 and 287; (d) LCDR1 comprising an amino acid sequence selected fromSEQ ID NOs: 129, 137, 145, 153, 161, 169, 177, 185, 193, 201, 209, 217,225, 233, 241, 249, 257, 265, 273, 281 and 289; (e) LCDR2 comprising anamino acid sequence selected from SEQ ID NOs: 130, 138, 146, 154, 162,170, 178, 186, 194, 202, 210, 218, 226, 234, 242, 250, 258, 266, 274,282, and 290; and (f) LCDR3 comprising an amino acid sequence selectedfrom SEQ ID NOs: 131, 139, 147, 155, 163, 171, 179, 187, 195, 203, 211,219, 227, 235, 243, 251, 259, 267, 275, 283 and 291.

In some embodiments, an anti-PD-1 antibody comprises at least one, two,three, four, five, or six CDRs selected from (a) HCDR1 comprising anamino acid sequence selected from SEQ ID NOs: 285, 237, 245, 253, 261and 269; (b) HCDR2 comprising an amino acid sequence selected from SEQID NOs: 286, 238, 246, 254, 262, and 270; (c) HCDR3 comprising an aminoacid sequence selected from SEQ ID NOs: 287, 239, 247, 255, 263, and271; (d) LCDR1 comprising an amino acid sequence selected from SEQ IDNOs: 289, 241, 249, 257, 265, and 273; (e) LCDR2 comprising an aminoacid sequence selected from SEQ ID NOs: 290, 242, 250, 258, 266, and274; and (f) LCDR3 comprising an amino acid sequence selected from SEQID NOs: 291, 243, 251, 259, 267, and 275.

In some embodiments, an anti-PD-1 antibody comprises at least one, two,three, four, five, or six CDRs selected from (a) HCDR1 comprising anamino acid sequence selected from SEQ ID NOs: 189, 197, 205, 213, 221,and 229; (b) HCDR2 comprising an amino acid sequence selected from SEQID NOs: 190, 198, 206, 214, 222, and 230; (c) HCDR3 comprising an aminoacid sequence selected from SEQ ID NOs: 191, 199, 207, 215, 223, and231; (d) LCDR1 comprising an amino acid sequence selected from SEQ IDNOs: 193, 201, 209, 217, 225, and 233; (e) LCDR2 comprising an aminoacid sequence selected from SEQ ID NOs: 194, 202, 210, 218, 226, and234; and (f) LCDR3 comprising an amino acid sequence selected from SEQID NOs: 195, 203, 211, 219, 227, and 235.

In some embodiments, an anti-PD-1 antibody comprises at least one, two,three, four, five, or six CDRs selected from (a) HCR1 comprising anamino acid sequence selected from SEQ ID NOs: 277, 125, 133, 141, 149,157, 165, 173, and 181; (b) HCDR2 comprising an amino acid sequenceselected from SEQ ID NOs: 278, 126, 134, 142, 150, 158, 166, 174, and182; (c) HCDR3 comprising an amino acid sequence selected from SEQ IDNOs: 279, 127, 135, 143, 151, 159, 167, 175, and 183; (d) LCDR1comprising an amino acid sequence selected from SEQ ID NOs: 281, 129,137, 145, 153, 161, 169, 177, and 185; (e) LCDR2 comprising an aminoacid sequence selected from SEQ ID NOs: 282, 130, 138, 146, 154, 162,170, 178, and 286; and (f) LCDR3 comprising an amino acid sequenceselected from SEQ ID NOs: 283, 131, 139, 147, 155, 163, 171, 179, and187.

In some embodiments, an anti-PD-1 antibody comprises at least one, two,three, four, five, or six CDRs selected from (a) HCDR1 comprising anamino acid sequence selected from SEQ ID NOs: 293, 301, 309 and 317; (b)HCDR2 comprising an amino acid sequence selected from SEQ ID NOs: 294,302, 310 and 318; (c) HCDR3 comprising an amino acid sequence selectedfrom SEQ ID NOs: 295, 303, 311 and 319; (d) LCDR1 comprising an aminoacid sequence selected from SEQ ID NOs: 297, 305, 313, and 321; (e)LCDR2 comprising an amino acid sequence selected from SEQ ID NOs: 298,306, 314 and 322; and (f) LCDR3 comprising an amino acid sequenceselected from SEQ ID NOs: 299, 307, 315, and 323.

In some embodiments, an anti-PD-1 antibody comprises a heavy chainvariable region and a light chain variable region. In some embodiments,an anti-PD-1 antibody comprises at least one heavy chain comprising aheavy chain variable region and at least a portion of a heavy chainconstant region, and at least one light chain comprising a light chainvariable region and at least a portion of a light chain constant region.In some embodiments, an anti-PD-1 antibody comprises two heavy chains,wherein each heavy chain comprises a heavy chain variable region and atleast a portion of a heavy chain constant region, and two light chains,wherein each light chain comprises a light chain variable region and atleast a portion of a light chain constant region. As used herein, asingle-chain Fv (scFv), or any other antibody that comprises, forexample, a single polypeptide chain comprising all six CDRs (three heavychain CDRs and three light chain CDRs) is considered to have a heavychain and a light chain. In some embodiments, the heavy chain is theregion of the anti-PD-1 antibody that comprises the three heavy chainCDRs. In some embodiments, the light chain is the region of theanti-PD-1 antibody that comprises the three light chain CDRs.

In some embodiments, the anti-PD-1 antibody comprises six CDRs including(a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 5; (b) HCDR2comprising the amino acid sequence of SEQ ID NO: 6; (c) HCDR3 comprisingthe amino acid sequence of SEQ ID NO: 7; (d) LCDR1 comprising the aminoacid sequence of SEQ ID NO: 9; (e) LCDR2 comprising the amino acidsequence of SEQ ID NO: 10; and (f) LCDR3 comprising the amino acidsequence of SEQ ID NO: 11.

In some embodiments, the anti-PD-1 antibody comprises six CDRs including(a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 13; (b) HCDR2comprising the amino acid sequence of SEQ ID NO: 14; (c) HCDR3comprising the amino acid sequence of SEQ ID NO: 15; (d) LCDR1comprising the amino acid sequence of SEQ ID NO: 17; (e) LCDR2comprising the amino acid sequence of SEQ ID NO: 18; and (f) LCDR3comprising the amino acid sequence of SEQ ID NO: 19.

In some embodiments, the anti-PD-1 antibody comprises six CDRs including(a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 21; (b) HCDR2comprising the amino acid sequence of SEQ ID NO: 22; (c) HCDR3comprising the amino acid sequence of SEQ ID NO: 23; (d) LCDR1comprising the amino acid sequence of SEQ ID NO: 25; (e) LCDR2comprising the amino acid sequence of SEQ ID NO: 26; and (f) LCDR3comprising the amino acid sequence of SEQ ID NO: 27.

In some embodiments, the anti-PD-1 antibody comprises six CDRs including(a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 29; (b) HCDR2comprising the amino acid sequence of SEQ ID NO: 30; (c) HCDR3comprising the amino acid sequence of SEQ ID NO: 31; (d) LCDR1comprising the amino acid sequence of SEQ ID NO: 33; (e) LCDR2comprising the amino acid sequence of SEQ ID NO: 34; and (f) LCDR3comprising the amino acid sequence of SEQ ID NO: 35.

In some embodiments, the anti-PD-1 antibody comprises six CDRs including(a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 37; (b) HCDR2comprising the amino acid sequence of SEQ ID NO: 38; (c) HCDR3comprising the amino acid sequence of SEQ ID NO: 39; (d) LCDR1comprising the amino acid sequence of SEQ ID NO: 41; (e) LCDR2comprising the amino acid sequence of SEQ ID NO: 42; and (f) LCDR3comprising the amino acid sequence of SEQ ID NO: 43.

In some embodiments, the anti-PD-1 antibody comprises six CDRs including(a) HCDR1 comprising an amino acid sequence selected from SEQ ID NOs:45, 53, 61, 69, 77, 85, 93, 101, 109 and 117; (b) HCDR2 comprising anamino acid sequence selected from SEQ ID NOs: 46, 54, 62, 70, 78, 86,94, 102, 110 and 118; (c) HCDR3 comprising an amino acid sequenceselected from SEQ ID NOs: 47, 55, 63, 71, 79, 87, 95, 103, 111, and 119;(d) LCDR1 comprising an amino acid sequence selected from SEQ ID NOs:49, 57, 65, 73, 81, 89, 97, 105, 113, and 121; (e) LCDR2 comprising anamino acid sequence selected from SEQ ID NOs: 50, 58, 66, 74, 82, 90,98, 106, 114, and 122; and (f) LCDR3 comprising an amino acid sequenceselected from SEQ ID NOs: 51, 59, 67, 75, 83, 91, 99, 107, 115, and 123.

In some embodiments, the anti-PD-1 antibody comprises six CDRs including(a) HCDR1 comprising an amino acid sequence selected from SEQ ID NOs:125, 133, 141, 149, 157, 165, 173, 181, 189, 197, 205, 213, 221, 229,237, 245, 253, 261, 269, 277 and 285; (b) HCDR2 comprising an amino acidsequence selected from SEQ ID NOs: 126, 134, 142, 150, 158, 166, 174,182, 190, 198, 206, 214, 222, 230, 238, 246, 254, 262, 270, 278 and 286;(c) HCDR3 comprising an amino acid sequence selected from SEQ ID NOs:127, 135, 143, 151, 159, 167, 175, 183, 191, 199, 207, 215, 223, 231,239, 247, 255, 263, 271, 279 and 287; (d) LCDR1 comprising an amino acidsequence selected from SEQ ID NOs: 129, 137, 145, 153, 161, 169, 177,185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265, 273, 281 and 289;(e) LCDR2 comprising an amino acid sequence selected from SEQ ID NOs:130, 138, 146, 154, 162, 170, 178, 186, 194, 202, 210, 218, 226, 234,242, 250, 258, 266, 274, 282, and 290; and (f) LCDR3 comprising an aminoacid sequence selected from SEQ ID NOs: 131, 139, 147, 155, 163, 171,179, 187, 195, 203, 211, 219, 227, 235, 243, 251, 259, 267, 275, 283 and291.

In some embodiments, the anti-PD-1 antibody comprises six CDRs including(a) HCDR1 comprising an amino acid sequence selected from SEQ ID NOs:285, 237, 245, 253, 261 and 269; (b) HCDR2 comprising an amino acidsequence selected from SEQ ID NOs: 286, 238, 246, 254, 262, and 270; (c)HCDR3 comprising an amino acid sequence selected from SEQ ID NOs: 287,239, 247, 255, 263, and 271; (d) LCDR1 comprising an amino acid sequenceselected from SEQ ID NOs: 289, 241, 249, 257, 265, and 273; (e) LCDR2comprising an amino acid sequence selected from SEQ ID NOs: 290, 242,250, 258, 266, and 274; and (f) LCDR3 comprising an amino acid sequenceselected from SEQ ID NOs: 291, 243, 251, 259, 267, and 275.

In some embodiments, the anti-PD-1 antibody comprises six CDRs including(a) HCDR1 comprising an amino acid sequence selected from SEQ ID NOs:189, 197, 205, 213, 221, and 229; (b) HCDR2 comprising an amino acidsequence selected from SEQ ID NOs: 190, 198, 206, 214, 222, and 230; (c)HCDR3 comprising an amino acid sequence selected from SEQ ID NOs: 191,199, 207, 215, 223, and 231; (d) LCDR1 comprising an amino acid sequenceselected from SEQ ID NOs: 193, 201, 209, 217, 225, and 233; (e) LCDR2comprising an amino acid sequence selected from SEQ ID NOs: 194, 202,210, 218, 226, and 234; and (f) LCDR3 comprising an amino acid sequenceselected from SEQ ID NOs: 195, 203, 211, 219, 227, and 235.

In some embodiments, the anti-PD-1 antibody comprises six CDRs including(a) HCR1 comprising an amino acid sequence selected from SEQ ID NOs:277, 125, 133, 141, 149, 157, 165, 173, and 181; (b) HCDR2 comprising anamino acid sequence selected from SEQ ID NOs: 278, 126, 134, 142, 150,158, 166, 174, and 182; (c) HCDR3 comprising an amino acid sequenceselected from SEQ ID NOs: 279, 127, 135, 143, 151, 159, 167, 175, and183; (d) LCDR1 comprising an amino acid sequence selected from SEQ IDNOs: 281, 129, 137, 145, 153, 161, 169, 177, and 185; (e) LCDR2comprising an amino acid sequence selected from SEQ ID NOs: 282, 130,138, 146, 154, 162, 170, 178, and 286; and (f) LCDR3 comprising an aminoacid sequence selected from SEQ ID NOs: 283, 131, 139, 147, 155, 163,171, 179, and 187.

In some embodiments, the anti-PD-1 antibody comprises six CDRs including(a) HCDR1 comprising an amino acid sequence selected from SEQ ID NOs:293, 301, 309 and 317; (b) HCDR2 comprising an amino acid sequenceselected from SEQ ID NOs: 294, 302, 310 and 318; (c) HCDR3 comprising anamino acid sequence selected from SEQ ID NOs: 295, 303, 311 and 319; (d)LCDR1 comprising an amino acid sequence selected from SEQ ID NOs: 297,305, 313, and 321; (e) LCDR2 comprising an amino acid sequence selectedfrom SEQ ID NOs: 298, 306, 314 and 322; and (f) LCDR3 comprising anamino acid sequence selected from SEQ ID NOs: 299, 307, 315, and 323.

In some embodiments, the anti-PD-1 antibody comprises the six CDRs asdescribed above and binds to PD-1. In some embodiments, the anti-PD-1antibody comprises the six CDRs as described above, binds to PD-1 andinhibits binding of PD-1 to PD-L1 and/or PD-L2. In some embodiments, theanti-PD-1 antibody comprises the six CDRs as described above, binds toPD-1 and inhibits binding of PD-1 to PD-L1. In some embodiments, theanti-PD-1 antibody comprises the six CDRs as described above, binds toPD-1 and inhibits binding of PD-1 to PD-L1 and PD-L2. In someembodiments, the anti-PD-1 antibody comprises the six CDRs as describedabove, binds to PD-1 and enhances an immune response in a subject,and/or increases activation of T cells in a subject followingadministration of the antibody to the subject.

In some embodiments, an anti-PD-1 antibody is provided that competeswith an anti-PD-1 antibody described herein for binding to PD-1. In someembodiments, an antibody that competes for binding with any of theantibodies provided herein can be made and/or used.

In some embodiments, the anti-PD-1 antibody comprises at least one, atleast two, or all three VH CDR sequences selected from (a) HCDR1comprising the amino acid sequence of SEQ ID NO: 5; (b) HCDR2 comprisingthe amino acid sequence of SEQ ID NO: 6; (c) HCDR3 comprising the aminoacid sequence of SEQ ID NO: 7.

In some embodiments, the anti-PD-1 antibody comprises at least one, atleast two, or all three VH CDR sequences selected from (a) HCDR1comprising the amino acid sequence of SEQ ID NO: 13; (b) HCDR2comprising the amino acid sequence of SEQ ID NO: 14; (c) HCDR3comprising the amino acid sequence of SEQ ID NO: 15.

In some embodiments, the anti-PD-1 antibody comprises at least one, atleast two, or all three VH CDR sequences selected from (a) HCDR1comprising the amino acid sequence of SEQ ID NO: 21; (b) HCDR2comprising the amino acid sequence of SEQ ID NO: 22; (c) HCDR3comprising the amino acid sequence of SEQ ID NO: 23.

In some embodiments, the anti-PD-1 antibody comprises at least one, atleast two, or all three VH CDR sequences selected from (a) HCDR1comprising the amino acid sequence of SEQ ID NO: 29; (b) HCDR2comprising the amino acid sequence of SEQ ID NO: 30; (c) HCDR3comprising the amino acid sequence of SEQ ID NO:31.

In some embodiments, the anti-PD-1 antibody comprises at least one, atleast two, or all three VH CDR sequences selected from (a) HCDR1comprising the amino acid sequence of SEQ ID NO: 37; (b) HCDR2comprising the amino acid sequence of SEQ ID NO: 38; (c) HCDR3comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, the anti-PD-1 antibody comprises at least one, atleast two, or all three VH CDR sequences selected from (a) HCDR1comprising the amino acid sequence of SEQ ID NO: 61; (b) HCDR2comprising the amino acid sequence of SEQ ID NO: 62; and (c) HCDR3comprising the amino acid sequence of SEQ ID NO: 63.

In some embodiments, the anti-PD-1 antibody comprises at least one, atleast two, or all three VH CDR sequences selected from (a) HCDR1comprising an amino acid sequence selected from SEQ ID NOs: 45, 53, 61,69, 77, 85, 93, 101, 109 and 117; (b) HCDR2 comprising an amino acidsequence selected from SEQ ID NOs: 46, 54, 62, 70, 78, 86, 94, 102, 110and 118; (c) HCDR3 comprising an amino acid sequence selected from SEQID NOs: 47, 55, 63, 71, 79, 87, 95, 103, 111, and 119.

In some embodiments, the anti-PD-1 antibody comprises at least one, atleast two, or all three VH CDR sequences selected from (a) HCDR1comprising an amino acid sequence selected from SEQ ID NOs: 125, 133,141, 149, 157, 165, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245,253, 261, 269, 277 and 285; (b) HCDR2 comprising an amino acid sequenceselected from SEQ ID NOs: 126, 134, 142, 150, 158, 166, 174, 182, 190,198, 206, 214, 222, 230, 238, 246, 254, 262, 270, 278 and 286; (c) HCDR3comprising an amino acid sequence selected from SEQ ID NOs: 127, 135,143, 151, 159, 167, 175, 183, 191, 199, 207, 215, 223, 231, 239, 247,255, 263, 271, 279 and 287.

In some embodiments, the anti-PD-1 antibody comprises at least one, atleast two, or all three VH CDR sequences selected from (a) HCDR1comprising an amino acid sequence selected from SEQ ID NOs: 285, 237,245, 253, 261 and 269; (b) HCDR2 comprising an amino acid sequenceselected from SEQ ID NOs: 286, 238, 246, 254, 262, and 270; (c) HCDR3comprising an amino acid sequence selected from SEQ ID NOs: 287, 239,247, 255, 263, and 271.

In some embodiments, the anti-PD-1 antibody comprises at least one, atleast two, or all three VH CDR sequences selected from (a) HCDR1comprising an amino acid sequence selected from SEQ ID NOs: 189, 197,205, 213, 221, and 229; (b) HCDR2 comprising an amino acid sequenceselected from SEQ ID NOs: 190, 198, 206, 214, 222, and 230; (c) HCDR3comprising an amino acid sequence selected from SEQ ID NOs: 191, 199,207, 215, 223, and 231.

In some embodiments, the anti-PD-1 antibody comprises at least one, atleast two, or all three VH CDR sequences selected from (a) HCR1comprising an amino acid sequence selected from SEQ ID NOs: 277, 125,133, 141, 149, 157, 165, 173, and 181; (b) HCDR2 comprising an aminoacid sequence selected from SEQ ID NOs: 278, 126, 134, 142, 150, 158,166, 174, and 182; (c) HCDR3 comprising an amino acid sequence selectedfrom SEQ ID NOs: 279, 127, 135, 143, 151, 159, 167, 175, and 183.

In some embodiments, the anti-PD-1 antibody comprises at least one, atleast two, or all three VH CDR sequences selected from (a) HCDR1comprising an amino acid sequence selected from SEQ ID NOs: 293, 301,309 and 317; (b) HCDR2 comprising an amino acid sequence selected fromSEQ ID NOs: 294, 302, 310 and 318; (c) HCDR3 comprising an amino acidsequence selected from SEQ ID NOs: 295, 303, 311 and 319.

In some embodiments, the antibody comprises at least one, at least two,or all three VL CDR sequences selected from (a) LCDR1 comprising theamino acid sequence of SEQ ID NO: 9; (b) LCDR2 comprising the amino acidsequence of SEQ ID NO: 10; and (c) LCDR3 comprising the amino acidsequence of SEQ ID NO: 11.

In some embodiments, the antibody comprises at least one, at least two,or all three VL CDR sequences selected from (a) LCDR1 comprising theamino acid sequence of SEQ ID NO: 17; (b) LCDR2 comprising the aminoacid sequence of SEQ ID NO: 18; and (c) LCDR3 comprising the amino acidsequence of SEQ ID NO: 19.

In some embodiments, the antibody comprises at least one, at least two,or all three VL CDR sequences selected from (a) LCDR1 comprising theamino acid sequence of SEQ ID NO: 25; (b) LCDR2 comprising the aminoacid sequence of SEQ ID NO: 26; and (c) LCDR3 comprising the amino acidsequence of SEQ ID NO: 27.

In some embodiments, the antibody comprises at least one, at least two,or all three VL CDR sequences selected from (a) LCDR1 comprising theamino acid sequence of SEQ ID NO: 33; (b) LCDR2 comprising the aminoacid sequence of SEQ ID NO: 34; and (c) LCDR3 comprising the amino acidsequence of SEQ ID NO: 35.

In some embodiments, the antibody comprises at least one, at least two,or all three VL CDR sequences selected from (a) LCDR1 comprising theamino acid sequence of SEQ ID NO: 41; (b) LCDR2 comprising the aminoacid sequence of SEQ ID NO: 42; and (c) LCDR3 comprising the amino acidsequence of SEQ ID NO: 43.

In some embodiments, the antibody comprises at least one, at least two,or all three VL CDR sequences selected from (a) LCDR1 comprising anamino acid sequence selected from SEQ ID NOs: 49, 57, 65, 73, 81, 89,97, 105, 113, and 121; (b) LCDR2 comprising an amino acid sequenceselected from SEQ ID NOs: 50, 58, 66, 74, 82, 90, 98, 106, 114, and 122;and (c) LCDR3 comprising an amino acid sequence selected from SEQ IDNOs: 51, 59, 67, 75, 83, 91, 99, 107, 115, and 123.

In some embodiments, the antibody comprises at least one, at least two,or all three VL CDR sequences selected from (a) LCDR1 comprising anamino acid sequence selected from SEQ ID NOs: 129, 137, 145, 153, 161,169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265, 273,281 and 289; (b) LCDR2 comprising an amino acid sequence selected fromSEQ ID NOs: 130, 138, 146, 154, 162, 170, 178, 186, 194, 202, 210, 218,226, 234, 242, 250, 258, 266, 274, 282, and 290; and (c) LCDR3comprising an amino acid sequence selected from SEQ ID NOs: 131, 139,147, 155, 163, 171, 179, 187, 195, 203, 211, 219, 227, 235, 243, 251,259, 267, 275, 283 and 291.

In some embodiments, the antibody comprises at least one, at least two,or all three VL CDR sequences selected from (a) LCDR1 comprising anamino acid sequence selected from SEQ ID NOs: 289, 241, 249, 257, 265,and 273; (b) LCDR2 comprising an amino acid sequence selected from SEQID NOs: 290, 242, 250, 258, 266, and 274; and (c) LCDR3 comprising anamino acid sequence selected from SEQ ID NOs: 291, 243, 251, 259, 267,and 275.

In some embodiments, the antibody comprises at least one, at least two,or all three VL CDR sequences selected (a) LCDR1 comprising an aminoacid sequence selected from SEQ ID NOs: 193, 201, 209, 217, 225, and233; (b) LCDR2 comprising an amino acid sequence selected from SEQ IDNOs: 194, 202, 210, 218, 226, and 234; and (c) LCDR3 comprising an aminoacid sequence selected from SEQ ID NOs: 195, 203, 211, 219, 227, and235.

In some embodiments, the antibody comprises at least one, at least two,or all three VL CDR sequences selected from (a) LCDR1 comprising anamino acid sequence selected from SEQ ID NOs: 281, 129, 137, 145, 153,161, 169, 177, and 185; (b) LCDR2 comprising an amino acid sequenceselected from SEQ ID NOs: 282, 130, 138, 146, 154, 162, 170, 178, and286; and (c) LCDR3 comprising an amino acid sequence selected from SEQID NOs: 283, 131, 139, 147, 155, 163, 171, 179, and 187.

In some embodiments, the antibody comprises at least one, at least two,or all three VL CDR sequences selected from (a) LCDR1 comprising anamino acid sequence selected from SEQ ID NOs: 297, 305, 313, and 321;(b) LCDR2 comprising an amino acid sequence selected from SEQ ID NOs:298, 306, 314 and 322; and (c) LCDR3 comprising an amino acid sequenceselected from SEQ ID NOs: 299, 307, 315, and 323.

In some embodiments, any of the six CDRs provided herein can be combinedas subparts with any of the other CDRs provided herein, for a total ofsix CDRs in a construct. Thus, in some embodiments, two CDRs from afirst antibody (for example, HCDR1 and HCDR2) can be combined with fourCDRs from a second antibody (HCDR3, LCDR1, LCDR2, and LCDR3). In someembodiments, two or fewer residues in one or more of the CDRs can bereplaced to obtain a variant thereof. In some embodiments, two or fewerresidues can be replaced in 1, 2, 3, 4, 5, or 6 of the CDRs.

In some embodiments, the anti-PD-1 antibody comprises (I) a VH domaincomprising at least one, at least two, or all three VH CDR sequencesselected from (a) HCDR1 comprising the amino acid sequence of SEQ ID NO:5; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO: 6; (c)HCDR3 comprising the amino acid sequence of SEQ ID NO: 7; and (II) a VLdomain comprising at least one, at least two, or all three VL CDRsequences selected from (d) LCDR1 comprising the amino acid sequence ofSEQ ID NO: 9; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO:10; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO: 11.

In some embodiments, the anti-PD-1 antibody comprises (I) a VH domaincomprising at least one, at least two, or all three VH CDR sequencesselected from (a) HCDR1 comprising the amino acid sequence of SEQ ID NO:13; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO: 14; (c)HCDR3 comprising the amino acid sequence of SEQ ID NO: 15; and (II) a VLdomain comprising at least one, at least two, or all three VL CDRsequences selected from (d) LCDR1 comprising the amino acid sequence ofSEQ ID NO: 17; (e) LCDR2 comprising the amino acid sequence of SEQ IDNO: 18; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO:19.

In some embodiments, the anti-PD-1 antibody comprises (I) a VH domaincomprising at least one, at least two, or all three VH CDR sequencesselected from (a) HCDR1 comprising the amino acid sequence of SEQ ID NO:21; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO: 22; (c)HCDR3 comprising the amino acid sequence of SEQ ID NO: 23; and (II) a VLdomain comprising at least one, at least two, or all three VL CDRsequences selected from (d) LCDR1 comprising the amino acid sequence ofSEQ ID NO: 25; (e) LCDR2 comprising the amino acid sequence of SEQ IDNO: 26; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO:27.

In some embodiments, the anti-PD-1 antibody comprises (I) a VH domaincomprising at least one, at least two, or all three VH CDR sequencesselected from (a) HCDR1 comprising the amino acid sequence of SEQ ID NO:29; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO: 30; (c)HCDR3 comprising the amino acid sequence of SEQ ID NO: 31; and (II) a VLdomain comprising at least one, at least two, or all three VL CDRsequences selected from (d) LCDR1 comprising the amino acid sequence ofSEQ ID NO: 33; (e) LCDR2 comprising the amino acid sequence of SEQ IDNO: 34; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO:35.

In some embodiments, the anti-PD-1 antibody comprises (I) a VH domaincomprising at least one, at least two, or all three VH CDR sequencesselected from (a) HCDR1 comprising the amino acid sequence of SEQ ID NO:37; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO: 38; (c)HCDR3 comprising the amino acid sequence of SEQ ID NO: 39; and (II) a VLdomain comprising at least one, at least two, or all three VL CDRsequences selected from (d) LCDR1 comprising the amino acid sequence ofSEQ ID NO: 41; (e) LCDR2 comprising the amino acid sequence of SEQ IDNO: 42; and (f) LCDR3 comprising the amino acid sequence of SEQ ID NO:43.

In some embodiments, the anti-PD-1 antibody comprises (I) a VH domaincomprising at least one, at least two, or all three VH CDR sequencesselected from (a) HCDR1 comprising an amino acid sequence selected fromSEQ ID NOs: 45, 53, 61, 69, 77, 85, 93, 101, 109 and 117; (b) HCDR2comprising an amino acid sequence selected from SEQ ID NOs: 46, 54, 62,70, 78, 86, 94, 102, 110 and 118; (c) HCDR3 comprising an amino acidsequence selected from SEQ ID NOs: 47, 55, 63, 71, 79, 87, 95, 103, 111,and 119; and (II) a VL domain comprising at least one, at least two, orall three VL CDR sequences selected from (d) LCDR1 comprising an aminoacid sequence selected from SEQ ID NOs: 49, 57, 65, 73, 81, 89, 97, 105,113, and 121; (e) LCDR2 comprising an amino acid sequence selected fromSEQ ID NOs: 50, 58, 66, 74, 82, 90, 98, 106, 114, and 122; and (f) LCDR3comprising an amino acid sequence selected from SEQ ID NOs: 51, 59, 67,75, 83, 91, 99, 107, 115, and 123.

In some embodiments, the anti-PD-1 antibody comprises (I) a VH domaincomprising at least one, at least two, or all three VH CDR sequencesselected from (a) HCDR1 comprising an amino acid sequence selected fromSEQ ID NOs: 125, 133, 141, 149, 157, 165, 173, 181, 189, 197, 205, 213,221, 229, 237, 245, 253, 261, 269, 277 and 285; (b) HCDR2 comprising anamino acid sequence selected from SEQ ID NOs: 126, 134, 142, 150, 158,166, 174, 182, 190, 198, 206, 214, 222, 230, 238, 246, 254, 262, 270,278 and 286; (c) HCDR3 comprising an amino acid sequence selected fromSEQ ID NOs: 127, 135, 143, 151, 159, 167, 175, 183, 191, 199, 207, 215,223, 231, 239, 247, 255, 263, 271, 279 and 287; and (II) a VL domaincomprising at least one, at least two, or all three VL CDR sequencesselected from (d) LCDR1 comprising an amino acid sequence selected fromSEQ ID NOs: 129, 137, 145, 153, 161, 169, 177, 185, 193, 201, 209, 217,225, 233, 241, 249, 257, 265, 273, 281 and 289; (e) LCDR2 comprising anamino acid sequence selected from SEQ ID NOs: 130, 138, 146, 154, 162,170, 178, 186, 194, 202, 210, 218, 226, 234, 242, 250, 258, 266, 274,282, and 290; and (f) LCDR3 comprising an amino acid sequence selectedfrom SEQ ID NOs: 131, 139, 147, 155, 163, 171, 179, 187, 195, 203, 211,219, 227, 235, 243, 251, 259, 267, 275, 283 and 291.

In some embodiments, the anti-PD-1 antibody comprises (I) a VH domaincomprising at least one, at least two, or all three VH CDR sequencesselected from (a) HCDR1 comprising an amino acid sequence selected fromSEQ ID NOs: 285, 237, 245, 253, 261 and 269; (b) HCDR2 comprising anamino acid sequence selected from SEQ ID NOs: 286, 238, 246, 254, 262,and 270; (c) HCDR3 comprising an amino acid sequence selected from SEQID NOs: 287, 239, 247, 255, 263, and 271; and (II) a VL domaincomprising at least one, at least two, or all three VL CDR sequencesselected from (d) LCDR1 comprising an amino acid sequence selected fromSEQ ID NOs: 289, 241, 249, 257, 265, and 273; (e) LCDR2 comprising anamino acid sequence selected from SEQ ID NOs: 290, 242, 250, 258, 266,and 274; and (f) LCDR3 comprising an amino acid sequence selected fromSEQ ID NOs: 291, 243, 251, 259, 267, and 275.

In some embodiments, the anti-PD-1 antibody comprises (I) a VH domaincomprising at least one, at least two, or all three VH CDR sequencesselected from (a) HCDR1 comprising an amino acid sequence selected fromSEQ ID NOs: 189, 197, 205, 213, 221, and 229; (b) HCDR2 comprising anamino acid sequence selected from SEQ ID NOs: 190, 198, 206, 214, 222,and 230; (c) HCDR3 comprising an amino acid sequence selected from SEQID NOs: 191, 199, 207, 215, 223, and 231; and (II) a VL domaincomprising at least one, at least two, or all three VL CDR sequencesselected from (d) LCDR1 comprising an amino acid sequence selected fromSEQ ID NOs: 193, 201, 209, 217, 225, and 233; (e) LCDR2 comprising anamino acid sequence selected from SEQ ID NOs: 194, 202, 210, 218, 226,and 234; and (f) LCDR3 comprising an amino acid sequence selected fromSEQ ID NOs: 195, 203, 211, 219, 227, and 235.

In some embodiments, the anti-PD-1 antibody comprises (I) a VH domaincomprising at least one, at least two, or all three VH CDR sequencesselected from (a) HCR1 comprising an amino acid sequence selected fromSEQ ID NOs: 277, 125, 133, 141, 149, 157, 165, 173, and 181; (b) HCDR2comprising an amino acid sequence selected from SEQ ID NOs: 278, 126,134, 142, 150, 158, 166, 174, and 182; (c) HCDR3 comprising an aminoacid sequence selected from SEQ ID NOs: 279, 127, 135, 143, 151, 159,167, 175, and 183; and (II) a VL domain comprising at least one, atleast two, or all three VL CDR sequences selected from (d) LCDR1comprising an amino acid sequence selected from SEQ ID NOs: 281, 129,137, 145, 153, 161, 169, 177, and 185; (e) LCDR2 comprising an aminoacid sequence selected from SEQ ID NOs: 282, 130, 138, 146, 154, 162,170, 178, and 286; and (f) LCDR3 comprising an amino acid sequenceselected from SEQ ID NOs: 283, 131, 139, 147, 155, 163, 171, 179, and187.

In some embodiments, the anti-PD-1 antibody comprises (I) a VH domaincomprising at least one, at least two, or all three VH CDR sequencesselected from (a) HCDR1 comprising an amino acid sequence selected fromSEQ ID NOs: 293, 301, 309 and 317; (b) HCDR2 comprising an amino acidsequence selected from SEQ ID NOs: 294, 302, 310 and 318; (c) HCDR3comprising an amino acid sequence selected from SEQ ID NOs: 295, 303,311 and 319; and (II) a VL domain comprising at least one, at least two,or all three VL CDR sequences selected from (d) LCDR1 comprising anamino acid sequence selected from SEQ ID NOs: 297, 305, 313, and 321;(e) LCDR2 comprising an amino acid sequence selected from SEQ ID NOs:298, 306, 314 and 322; and (f) LCDR3 comprising an amino acid sequenceselected from SEQ ID NOs: 299, 307, 315, and 323.

In some embodiments, an anti-PD-1 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92,100, 108, 116, 124, 132, 140, 148, 156, 164, 172, 180, 188, 196, 204,212, 220, 228, 236, 244, 252, 260, 268, 276, 284, 292, 300, 308, or 316.In some embodiments, a VH sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (forexample, conservative substitutions), insertions, or deletions relativeto the reference sequence, but an anti-PD-1 antibody comprising thatsequence retains the ability to bind to PD-1. In some embodiments, atotal of 1 to 10 amino acids have been substituted, inserted and/ordeleted in SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92,100, 108, 116, 124, 132, 140, 148, 156, 164, 172, 180, 188, 196, 204,212, 220, 228, 236, 244, 252, 260, 268, 276, 284, 292, 300, 308, or 316.In some embodiments, substitutions, insertions, or deletions occur inregions outside the CDRs (that is, in the FRs). Optionally, theanti-PD-1 antibody comprises the VH sequence in SEQ ID NO: 4, 12, 20,28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108, 116, 124, 132, 140, 148,156, 164, 172, 180, 188, 196, 204, 212, 220, 228, 236, 244, 252, 260,268, 276, 284, 292, 300, 308, or 316, including post-translationalmodifications of that sequence.

In some embodiments, the VH comprises: (a) HCDR1 comprising the aminoacid sequence of SEQ ID NO: 5; (b) HCDR2 comprising the amino acidsequence of SEQ ID NO: 6; (c) HCDR3 comprising the amino acid sequenceof SEQ ID NO: 7.

In some embodiments, the VH comprises: (a) HCDR1 comprising the aminoacid sequence of SEQ ID NO: 13; (b) HCDR2 comprising the amino acidsequence of SEQ ID NO: 14; (c) HCDR3 comprising the amino acid sequenceof SEQ ID NO: 15.

In some embodiments, the VH comprises: (a) HCDR1 comprising the aminoacid sequence of SEQ ID NO: 21; (b) HCDR2 comprising the amino acidsequence of SEQ ID NO: 22; (c) HCDR3 comprising the amino acid sequenceof SEQ ID NO: 23.

In some embodiments, the VH comprises: (a) HCDR1 comprising the aminoacid sequence of SEQ ID NO: 29; (b) HCDR2 comprising the amino acidsequence of SEQ ID NO: 30; (c) HCDR3 comprising the amino acid sequenceof SEQ ID NO: 31.

In some embodiments, the VH comprises: (a) HCDR1 comprising the aminoacid sequence of SEQ ID NO: 37; (b) HCDR2 comprising the amino acidsequence of SEQ ID NO: 38; (c) HCDR3 comprising the amino acid sequenceof SEQ ID NO: 39.

In some embodiments, the VH comprises: (a) HCDR1 comprising an aminoacid sequence selected from SEQ ID NOs: 45, 53, 61, 69, 77, 85, 93, 101,109 and 117; (b) HCDR2 comprising an amino acid sequence selected fromSEQ ID NOs: 46, 54, 62, 70, 78, 86, 94, 102, 110 and 118; (c) HCDR3comprising an amino acid sequence selected from SEQ ID NOs: 47, 55, 63,71, 79, 87, 95, 103, 111, and 119.

In some embodiments, the VH comprises: (a) HCDR1 comprising an aminoacid sequence selected from SEQ ID NOs: 125, 133, 141, 149, 157, 165,173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, 269, 277 and285; (b) HCDR2 comprising an amino acid sequence selected from SEQ IDNOs: 126, 134, 142, 150, 158, 166, 174, 182, 190, 198, 206, 214, 222,230, 238, 246, 254, 262, 270, 278 and 286; (c) HCDR3 comprising an aminoacid sequence selected from SEQ ID NOs: 127, 135, 143, 151, 159, 167,175, 183, 191, 199, 207, 215, 223, 231, 239, 247, 255, 263, 271, 279 and287.

In some embodiments, the VH comprises: (a) HCDR1 comprising an aminoacid sequence selected from SEQ ID NOs: 285, 237, 245, 253, 261 and 269;(b) HCDR2 comprising an amino acid sequence selected from SEQ ID NOs:286, 238, 246, 254, 262, and 270; (c) HCDR3 comprising an amino acidsequence selected from SEQ ID NOs: 287, 239, 247, 255, 263, and 271.

In some embodiments, the VH comprises: (a) HCDR1 comprising an aminoacid sequence selected from SEQ ID NOs: 189, 197, 205, 213, 221, and229; (b) HCDR2 comprising an amino acid sequence selected from SEQ IDNOs: 190, 198, 206, 214, 222, and 230; (c) HCDR3 comprising an aminoacid sequence selected from SEQ ID NOs: 191, 199, 207, 215, 223, and231.

In some embodiments, the VH comprises: (a) HCR1 comprising an amino acidsequence selected from SEQ ID NOs: 277, 125, 133, 141, 149, 157, 165,173, and 181; (b) HCDR2 comprising an amino acid sequence selected fromSEQ ID NOs: 278, 126, 134, 142, 150, 158, 166, 174, and 182; (c) HCDR3comprising an amino acid sequence selected from SEQ ID NOs: 279, 127,135, 143, 151, 159, 167, 175, and 183.

In some embodiments, the VH comprises: (a) HCDR1 comprising an aminoacid sequence selected from SEQ ID NOs: 293, 301, 309 and 317; (b) HCDR2comprising an amino acid sequence selected from SEQ ID NOs: 294, 302,310 and 318; (c) HCDR3 comprising an amino acid sequence selected fromSEQ ID NOs: 295, 303, 311 and 319.

In some embodiments, an anti-PD-1 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 8, 16, 24, 32, 40, 48,56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 160, 168,176, 184, 192, 200, 208, 216, 224, 232, 240, 248, 256, 264, 272, 280,288, 296, 304, 312 or 320. In some embodiments, a VL sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identitycontains substitutions (for example, conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-PD-1 antibody comprising that sequence retains the ability to bindto PD-1. In some embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO: 8, 16, 24, 32, 40,48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 160, 168,176, 184, 192, 200, 208, 216, 224, 232, 240, 248, 256, 264, 272, 280,288, 296, 304, 312 or 320. In some embodiments, the substitutions,insertions, or deletions occur in regions outside the CDRs (that is, inthe FRs). Optionally, the anti-PD-1 antibody comprises the VL sequencein SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112,120, 128, 136, 144, 152, 160, 168, 176, 184, 192, 200, 208, 216, 224,232, 240, 248, 256, 264, 272, 280, 288, 296, 304, 312 or 320, includingpost-translational modifications of that sequence.

In some embodiments, the VL comprises: (a) LCDR1 comprising the aminoacid sequence of SEQ ID NO: 9; (b) LCDR2 comprising the amino acidsequence of SEQ ID NO: 10; and (c) LCDR3 comprising the amino acidsequence of SEQ ID NO: 11.

In some embodiments, the VL comprises: (a) LCDR1 comprising the aminoacid sequence of SEQ ID NO: 17; (b) LCDR2 comprising the amino acidsequence of SEQ ID NO: 18; and (c) LCDR3 comprising the amino acidsequence of SEQ ID NO: 19.

In some embodiments, the VL comprises: (a) LCDR1 comprising the aminoacid sequence of SEQ ID NO: 25; (b) LCDR2 comprising the amino acidsequence of SEQ ID NO: 26; and (c) LCDR3 comprising the amino acidsequence of SEQ ID NO: 27.

In some embodiments, the VL comprises: (a) LCDR1 comprising the aminoacid sequence of SEQ ID NO: 33; (b) LCDR2 comprising the amino acidsequence of SEQ ID NO: 34; and (c) LCDR3 comprising the amino acidsequence of SEQ ID NO: 35.

In some embodiments, the VL comprises: (a) LCDR1 comprising the aminoacid sequence of SEQ ID NO: 41; (b) LCDR2 comprising the amino acidsequence of SEQ ID NO: 42; and (c) LCDR3 comprising the amino acidsequence of SEQ ID NO: 43.

In some embodiments, the VL comprises: (a) LCDR1 comprising an aminoacid sequence selected from SEQ ID NOs: 49, 57, 65, 73, 81, 89, 97, 105,113, and 121; (b) LCDR2 comprising an amino acid sequence selected fromSEQ ID NOs: 50, 58, 66, 74, 82, 90, 98, 106, 114, and 122; and (c) LCDR3comprising an amino acid sequence selected from SEQ ID NOs: 51, 59, 67,75, 83, 91, 99, 107, 115, and 123.

In some embodiments, the VL comprises: (a) LCDR1 comprising an aminoacid sequence selected from SEQ ID NOs: 129, 137, 145, 153, 161, 169,177, 185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265, 273, 281 and289; (b) LCDR2 comprising an amino acid sequence selected from SEQ IDNOs: 130, 138, 146, 154, 162, 170, 178, 186, 194, 202, 210, 218, 226,234, 242, 250, 258, 266, 274, 282, and 290; and (c) LCDR3 comprising anamino acid sequence selected from SEQ ID NOs: 131, 139, 147, 155, 163,171, 179, 187, 195, 203, 211, 219, 227, 235, 243, 251, 259, 267, 275,283 and 291.

In some embodiments, the VL comprises: (a) LCDR1 comprising an aminoacid sequence selected from SEQ ID NOs: 289, 241, 249, 257, 265, and273; (b) LCDR2 comprising an amino acid sequence selected from SEQ IDNOs: 290, 242, 250, 258, 266, and 274; and (c) LCDR3 comprising an aminoacid sequence selected from SEQ ID NOs: 291, 243, 251, 259, 267, and275.

In some embodiments, the VL comprises: (a) LCDR1 comprising an aminoacid sequence selected from SEQ ID NOs: 193, 201, 209, 217, 225, and233; (b) LCDR2 comprising an amino acid sequence selected from SEQ IDNOs: 194, 202, 210, 218, 226, and 234; and (c) LCDR3 comprising an aminoacid sequence selected from SEQ ID NOs: 195, 203, 211, 219, 227, and235.

In some embodiments, the VL comprises: (a) LCDR1 comprising an aminoacid sequence selected from SEQ ID NOs: 281, 129, 137, 145, 153, 161,169, 177, and 185; (b) LCDR2 comprising an amino acid sequence selectedfrom SEQ ID NOs: 282, 130, 138, 146, 154, 162, 170, 178, and 286; and(c) LCDR3 comprising an amino acid sequence selected from SEQ ID NOs:283, 131, 139, 147, 155, 163, 171, 179, and 187.

In some embodiments, the VL comprises: (a) LCDR1 comprising an aminoacid sequence selected from SEQ ID NOs: 297, 305, 313, and 321; (b)LCDR2 comprising an amino acid sequence selected from SEQ ID NOs: 298,306, 314 and 322; and (c) LCDR3 comprising an amino acid sequenceselected from SEQ ID NOs: 299, 307, 315, and 323.

In some embodiments, an anti-PD-1 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92,100, 108, 116, 124, 132, 140, 148, 156, 164, 172, 180, 188, 196, 204,212, 220, 228, 236, 244, 252, 260, 268, 276, 284, 292, 300, 308, or 316and a light chain variable domain (VL) having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80,88, 96, 104, 112, 120, 128, 136, 144, 152, 160, 168, 176, 184, 192, 200,208, 216, 224, 232, 240, 248, 256, 264, 272, 280, 288, 296, 304, 312 or320. In some embodiments, a VH sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions(for example, conservative substitutions), insertions, or deletionsrelative to the reference sequence, and a VL sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (for example, conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-PD-1 antibodycomprising that sequence retains the ability to bind to PD-1. In someembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68,76, 84, 92, 100, 108, 116, 124, 132, 140, 148, 156, 164, 172, 180, 188,196, 204, 212, 220, 228, 236, 244, 252, 260, 268, 276, 284, 292, 300,308, or 316. In some embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO: 8, 16, 24, 32,40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 160,168, 176, 184, 192, 200, 208, 216, 224, 232, 240, 248, 256, 264, 272,280, 288, 296, 304, 312 or 320. In some embodiments, substitutions,insertions, or deletions occur in regions outside the CDRs (that is, inthe FRs). Optionally, the anti-PD-1 antibody comprises the VH sequencein SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108,116, 124, 132, 140, 148, 156, 164, 172, 180, 188, 196, 204, 212, 220,228, 236, 244, 252, 260, 268, 276, 284, 292, 300, 308, or 316 and the VLsequence of SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96,104, 112, 120, 128, 136, 144, 152, 160, 168, 176, 184, 192, 200, 208,216, 224, 232, 240, 248, 256, 264, 272, 280, 288, 296, 304, 312 or 320,including post-translational modifications of one or both sequence.

In some embodiments, the anti-PD-1 antibody comprises (a) HCDR1comprising the amino acid sequence of SEQ ID NO: 5; (b) HCDR2 comprisingthe amino acid sequence of SEQ ID NO: 6; (c) HCDR3 comprising the aminoacid sequence of SEQ ID NO: 7; (d) LCDR1 comprising the amino acidsequence of SEQ ID NO: 9; (e) LCDR2 comprising the amino acid sequenceof SEQ ID NO: 10; and (f) LCDR3 comprising the amino acid sequence ofSEQ ID NO: 11.

In some embodiments, the anti-PD-1 antibody comprises (a) HCDR1comprising the amino acid sequence of SEQ ID NO: 13; (b) HCDR2comprising the amino acid sequence of SEQ ID NO: 14; (c) HCDR3comprising the amino acid sequence of SEQ ID NO: 15; (d) LCDR1comprising the amino acid sequence of SEQ ID NO: 17; (e) LCDR2comprising the amino acid sequence of SEQ ID NO: 18; and (f) LCDR3comprising the amino acid sequence of SEQ ID NO: 19.

In some embodiments, the anti-PD-1 antibody comprises (a) HCDR1comprising the amino acid sequence of SEQ ID NO: 21; (b) HCDR2comprising the amino acid sequence of SEQ ID NO: 22; (c) HCDR3comprising the amino acid sequence of SEQ ID NO: 23; (d) LCDR1comprising the amino acid sequence of SEQ ID NO: 25; (e) LCDR2comprising the amino acid sequence of SEQ ID NO: 26; and (f) LCDR3comprising the amino acid sequence of SEQ ID NO: 27.

In some embodiments, the anti-PD-1 antibody comprises (a) HCDR1comprising the amino acid sequence of SEQ ID NO: 29; (b) HCDR2comprising the amino acid sequence of SEQ ID NO: 30; (c) HCDR3comprising the amino acid sequence of SEQ ID NO: 31; (d) LCDR1comprising the amino acid sequence of SEQ ID NO: 33; (e) LCDR2comprising the amino acid sequence of SEQ ID NO: 34; and (f) LCDR3comprising the amino acid sequence of SEQ ID NO: 35.

In some embodiments, the anti-PD-1 antibody comprises (a) HCDR1comprising the amino acid sequence of SEQ ID NO: 37; (b) HCDR2comprising the amino acid sequence of SEQ ID NO: 38; (c) HCDR3comprising the amino acid sequence of SEQ ID NO: 39; (d) LCDR1comprising the amino acid sequence of SEQ ID NO: 41; (e) LCDR2comprising the amino acid sequence of SEQ ID NO: 42; and (f) LCDR3comprising the amino acid sequence of SEQ ID NO: 43.

In some embodiments, the anti-PD-1 antibody comprises (a) HCDR1comprising an amino acid sequence selected from SEQ ID NOs: 45, 53, 61,69, 77, 85, 93, 101, 109 and 117; (b) HCDR2 comprising an amino acidsequence selected from SEQ ID NOs: 46, 54, 62, 70, 78, 86, 94, 102, 110and 118; (c) HCDR3 comprising an amino acid sequence selected from SEQID NOs: 47, 55, 63, 71, 79, 87, 95, 103, 111, and 119; (d) LCDR1comprising an amino acid sequence selected from SEQ ID NOs: 49, 57, 65,73, 81, 89, 97, 105, 113, and 121; (e) LCDR2 comprising an amino acidsequence selected from SEQ ID NOs: 50, 58, 66, 74, 82, 90, 98, 106, 114,and 122; and (f) LCDR3 comprising an amino acid sequence selected fromSEQ ID NOs: 51, 59, 67, 75, 83, 91, 99, 107, 115, and 123.

In some embodiments, the anti-PD-1 antibody comprises (a) HCDR1comprising an amino acid sequence selected from SEQ ID NOs: 125, 133,141, 149, 157, 165, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245,253, 261, 269, 277 and 285; (b) HCDR2 comprising an amino acid sequenceselected from SEQ ID NOs: 126, 134, 142, 150, 158, 166, 174, 182, 190,198, 206, 214, 222, 230, 238, 246, 254, 262, 270, 278 and 286; (c) HCDR3comprising an amino acid sequence selected from SEQ ID NOs: 127, 135,143, 151, 159, 167, 175, 183, 191, 199, 207, 215, 223, 231, 239, 247,255, 263, 271, 279 and 287; (d) LCDR1 comprising an amino acid sequenceselected from SEQ ID NOs: 129, 137, 145, 153, 161, 169, 177, 185, 193,201, 209, 217, 225, 233, 241, 249, 257, 265, 273, 281 and 289; (e) LCDR2comprising an amino acid sequence selected from SEQ ID NOs: 130, 138,146, 154, 162, 170, 178, 186, 194, 202, 210, 218, 226, 234, 242, 250,258, 266, 274, 282, and 290; and (f) LCDR3 comprising an amino acidsequence selected from SEQ ID NOs: 131, 139, 147, 155, 163, 171, 179,187, 195, 203, 211, 219, 227, 235, 243, 251, 259, 267, 275, 283 and 291.

In some embodiments, the anti-PD-1 antibody comprises (a) HCDR1comprising an amino acid sequence selected from SEQ ID NOs: 285, 237,245, 253, 261 and 269; (b) HCDR2 comprising an amino acid sequenceselected from SEQ ID NOs: 286, 238, 246, 254, 262, and 270; (c) HCDR3comprising an amino acid sequence selected from SEQ ID NOs: 287, 239,247, 255, 263, and 271; (d) LCDR1 comprising an amino acid sequenceselected from SEQ ID NOs: 289, 241, 249, 257, 265, and 273; (e) LCDR2comprising an amino acid sequence selected from SEQ ID NOs: 290, 242,250, 258, 266, and 274; and (f) LCDR3 comprising an amino acid sequenceselected from SEQ ID NOs: 291, 243, 251, 259, 267, and 275.

In some embodiments, the anti-PD-1 antibody comprises (a) HCDR1comprising an amino acid sequence selected from SEQ ID NOs: 189, 197,205, 213, 221, and 229; (b) HCDR2 comprising an amino acid sequenceselected from SEQ ID NOs: 190, 198, 206, 214, 222, and 230; (c) HCDR3comprising an amino acid sequence selected from SEQ ID NOs: 191, 199,207, 215, 223, and 231; (d) LCDR1 comprising an amino acid sequenceselected from SEQ ID NOs: 193, 201, 209, 217, 225, and 233; (e) LCDR2comprising an amino acid sequence selected from SEQ ID NOs: 194, 202,210, 218, 226, and 234; and (f) LCDR3 comprising an amino acid sequenceselected from SEQ ID NOs: 195, 203, 211, 219, 227, and 235.

In some embodiments, the anti-PD-1 antibody comprises (a) HCR1comprising an amino acid sequence selected from SEQ ID NOs: 277, 125,133, 141, 149, 157, 165, 173, and 181; (b) HCDR2 comprising an aminoacid sequence selected from SEQ ID NOs: 278, 126, 134, 142, 150, 158,166, 174, and 182; (c) HCDR3 comprising an amino acid sequence selectedfrom SEQ ID NOs: 279, 127, 135, 143, 151, 159, 167, 175, and 183; (d)LCDR1 comprising an amino acid sequence selected from SEQ ID NOs: 281,129, 137, 145, 153, 161, 169, 177, and 185; (e) LCDR2 comprising anamino acid sequence selected from SEQ ID NOs: 282, 130, 138, 146, 154,162, 170, 178, and 286; and (f) LCDR3 comprising an amino acid sequenceselected from SEQ ID NOs: 283, 131, 139, 147, 155, 163, 171, 179, and187.

In some embodiments, the anti-PD-1 antibody comprises (a) HCDR1comprising an amino acid sequence selected from SEQ ID NOs: 293, 301,309 and 317; (b) HCDR2 comprising an amino acid sequence selected fromSEQ ID NOs: 294, 302, 310 and 318; (c) HCDR3 comprising an amino acidsequence selected from SEQ ID NOs: 295, 303, 311 and 319; (d) LCDR1comprising an amino acid sequence selected from SEQ ID NOs: 297, 305,313, and 321; (e) LCDR2 comprising an amino acid sequence selected fromSEQ ID NOs: 298, 306, 314 and 322; and (f) LCDR3 comprising an aminoacid sequence selected from SEQ ID NOs: 299, 307, 315, and 323.

In some embodiments, an anti-PD-1 antibody comprises a VH as in any ofthe embodiments provided herein, and a VL as in any of the embodimentsprovided herein. In some embodiments, the antibody comprises the VH andVL sequences in SEQ ID NO: 4 and SEQ ID NO: 8, respectively, includingpost-translational modifications of those sequences. In someembodiments, the antibody comprises the VH and VL sequences in SEQ IDNO: 12 and SEQ ID NO: 16, respectively, including post-translationalmodifications of those sequences. In some embodiments, the antibodycomprises the VH and VL sequences in SEQ ID NO: 20 and SEQ ID NO: 24,respectively, including post-translational modifications of thosesequences. In some embodiments, the antibody comprises the VH and VLsequences in SEQ ID NO: 28 and SEQ ID NO: 32, respectively, includingpost-translational modifications of those sequences. In someembodiments, the antibody comprises the VH and VL sequences in SEQ IDNO: 36 and SEQ ID NO: 40, respectively, including post-translationalmodifications of those sequences. In some embodiments, the antibodycomprises the VH and VL sequences in SEQ ID NO: 44 and SEQ ID NO: 48,respectively, including post-translational modifications of thosesequences. In some embodiments, the antibody comprises the VH and VLsequences in SEQ ID NO: 52 and SEQ ID NO: 56, respectively, includingpost-translational modifications of those sequences. In someembodiments, the antibody comprises the VH and VL sequences in SEQ IDNO: 60 and SEQ ID NO: 64, respectively, including post-translationalmodifications of those sequences. In some embodiments, the antibodycomprises the VH and VL sequences in SEQ ID NO: 68 and SEQ ID NO: 72,respectively, including post-translational modifications of thosesequences. In some embodiments, the antibody comprises the VH and VLsequences in SEQ ID NO: 76 and SEQ ID NO: 80, respectively, includingpost-translational modifications of those sequences. In someembodiments, the antibody comprises the VH and VL sequences in SEQ IDNO: 84 and SEQ ID NO: 88, respectively, including post-translationalmodifications of those sequences. In some embodiments, the antibodycomprises the VH and VL sequences in SEQ ID NO: 92 and SEQ ID NO: 96,respectively, including post-translational modifications of thosesequences. In some embodiments, the antibody comprises the VH and VLsequences in SEQ ID NO: 100 and SEQ ID NO: 104, respectively, includingpost-translational modifications of those sequences. In someembodiments, the antibody comprises the VH and VL sequences in SEQ IDNO: 108 and SEQ ID NO: 112, respectively, including post-translationalmodifications of those sequences. In some embodiments, the antibodycomprises the VH and VL sequences in SEQ ID NO: 116 and SEQ ID NO: 120,respectively, including post-translational modifications of thosesequences. In some embodiments, the antibody comprises the VH and VLsequences in SEQ ID NO: 124 and SEQ ID NO: 128, respectively, includingpost-translational modifications of those sequences. In someembodiments, the antibody comprises the VH and VL sequences in SEQ IDNO: 132 and SEQ ID NO: 136, respectively, including post-translationalmodifications of those sequences. In some embodiments, the antibodycomprises the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 144,respectively, including post-translational modifications of thosesequences. In some embodiments, the antibody comprises the VH and VLsequences in SEQ ID NO: 148 and SEQ ID NO: 152, respectively, includingpost-translational modifications of those sequences. In someembodiments, the antibody comprises the VH and VL sequences in SEQ IDNO: 156 and SEQ ID NO: 160, respectively, including post-translationalmodifications of those sequences. In some embodiments, the antibodycomprises the VH and VL sequences in SEQ ID NO: 164 and SEQ ID NO: 168,respectively, including post-translational modifications of thosesequences. In some embodiments, the antibody comprises the VH and VLsequences in SEQ ID NO: 172 and SEQ ID NO: 176, respectively, includingpost-translational modifications of those sequences. In someembodiments, the antibody comprises the VH and VL sequences in SEQ IDNO: 180 and SEQ ID NO: 184, respectively, including post-translationalmodifications of those sequences. In some embodiments, the antibodycomprises the VH and VL sequences in SEQ ID NO: 188 and SEQ ID NO: 192,respectively, including post-translational modifications of thosesequences. In some embodiments, the antibody comprises the VH and VLsequences in SEQ ID NO: 196 and SEQ ID NO: 200, respectively, includingpost-translational modifications of those sequences. In someembodiments, the antibody comprises the VH and VL sequences in SEQ IDNO: 204 and SEQ ID NO: 208, respectively, including post-translationalmodifications of those sequences. In some embodiments, the antibodycomprises the VH and VL sequences in SEQ ID NO: 212 and SEQ ID NO: 216,respectively, including post-translational modifications of thosesequences. In some embodiments, the antibody comprises the VH and VLsequences in SEQ ID NO: 220 and SEQ ID NO: 224, respectively, includingpost-translational modifications of those sequences. In someembodiments, the antibody comprises the VH and VL sequences in SEQ IDNO: 228 and SEQ ID NO: 232, respectively, including post-translationalmodifications of those sequences. In some embodiments, the antibodycomprises the VH and VL sequences in SEQ ID NO: 236 and SEQ ID NO: 240,respectively, including post-translational modifications of thosesequences. In some embodiments, the antibody comprises the VH and VLsequences in SEQ ID NO: 244 and SEQ ID NO: 248, respectively, includingpost-translational modifications of those sequences. In someembodiments, the antibody comprises the VH and VL sequences in SEQ IDNO: 252 and SEQ ID NO: 256, respectively, including post-translationalmodifications of those sequences. In some embodiments, the antibodycomprises the VH and VL sequences in SEQ ID NO: 260 and SEQ ID NO: 264,respectively, including post-translational modifications of thosesequences. In some embodiments, the antibody comprises the VH and VLsequences in SEQ ID NO: 268 and SEQ ID NO: 272, respectively, includingpost-translational modifications of those sequences. In someembodiments, the antibody comprises the VH and VL sequences in SEQ IDNO: 276 and SEQ ID NO: 280, respectively, including post-translationalmodifications of those sequences. In some embodiments, the antibodycomprises the VH and VL sequences in SEQ ID NO: 284 and SEQ ID NO: 288,respectively, including post-translational modifications of thosesequences. In some embodiments, the antibody comprises the VH and VLsequences in SEQ ID NO: 292 and SEQ ID NO: 296, respectively, includingpost-translational modifications of those sequences. In someembodiments, the antibody comprises the VH and VL sequences in SEQ IDNO: 300 and SEQ ID NO: 304, respectively, including post-translationalmodifications of those sequences. In some embodiments, the antibodycomprises the VH and VL sequences in SEQ ID NO: 308 and SEQ ID NO: 312,respectively, including post-translational modifications of thosesequences. In some embodiments, the antibody comprises the VH and VLsequences in SEQ ID NO: 316 and SEQ ID NO: 320, respectively, includingpost-translational modifications of those sequences.

In some embodiments, antibodies which compete with the anti-PD-1antibodies provided herein for binding to PD-1 are provided. In someembodiments, antibodies compete with the anti-PD-1 antibodies providedherein for binding to an epitope on PD-1.

In some embodiments, competition assays may be used to identify amonoclonal antibody that competes with an anti-PD-1 antibody describedherein (such as 12228, 13406, 13407, 13408, and 13409) for binding toPD-1. Competition assays can be used to determine whether two antibodiesbind the same epitope by recognizing identical or sterically overlappingepitopes or one antibody competitively inhibits binding of anotherantibody to the antigen. In some embodiments, such a competing antibodybinds to the same epitope that is bound by an antibody described herein.Exemplary competition assays include, but are not limited to, routineassays such as those provided in Harlow and Lane (1988) Antibodies: ALaboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.). Detailed exemplary methods for mapping an epitope towhich an antibody binds are provided in Morris (1996) “Epitope MappingProtocols,” in Methods in Molecular Biology vol. 66 (Humana Press,Totowa, N.J.). In some embodiments, two antibodies are said to bind tothe same epitope if each blocks binding of the other by 50% or more. Insome embodiments, the antibody that competes with an anti-PD-1 antibodydescribed herein is a chimeric, humanized or human antibody. In someembodiments, an antibody that competes with a chimeric, humanized, orhuman anti-PD-1 antibody as described herein is provided.

In some embodiments, antibodies that bind to any one or more of theepitopes that the antibodies provided herein are provided. In someembodiments, antibodies that bind and overlap an epitope to which thepresent antibodies bind to are provided. In some embodiments, anantibody is provided that competes with at least one of the antibodiesprovided herein. In some embodiments, an antibody is provided thatcompetes with at least two of the antibodies provided herein. In someembodiments, an antibody is provided that competes with at least threeof the antibodies provided herein. In some embodiments, the antibodybinds to an overlapping epitope as an antibody described in the examplesherein. In some embodiments, the entire epitope is bound and/orobstructed by the competing antibody. In some embodiments, a part of theepitope is bound and/or obstructed by the competing antibody. In someembodiments, the competing antibody's paratope binds to at least a partof the epitope of an antibody provided herein. In some embodiments, thecompeting antibody's paratope binds the target, and a different sectionof the competing antibody's structure obstruct at least a part of theepitope of an antibody provided herein.

Exemplary Chimeric Antibodies

In some embodiments, an antibody provided herein is a chimeric antibody.Certain chimeric antibodies are described, for example, in U.S. Pat. No.4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (for example, a variable region derived from amouse, rat, hamster, rabbit, or non-human primate, such as a monkey) anda human constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

Nonlimiting exemplary chimeric antibodies include chimeric antibodiescomprising the heavy and/or light chain variable regions of an antibodyselected from, e.g., antibody 11606; antibody 11613; antibody 11645;antibody 12191; antibody 12195; antibody 12220; antibody 12228; antibody12535; antibody 12536; antibody 12541; antibody 12543; antibody 12544;antibody 12545; antibody 12549; antibody 12550; antibody 12553; antibody12554; antibody 12562; antibody 12563; antibody 12564; antibody 12565;antibody 12571; antibody 12572; antibody 12576; antibody 12583; antibody12584; antibody 13396; antibody 13398; antibody 13399; antibody 13401;antibody 13402; antibody 13403; antibody 13404; antibody 13405; antibody13406; antibody 13407; antibody 13408; antibody 13409; antibody 11624;and antibody 12190, as disclosed herein. Additional nonlimitingexemplary chimeric antibodies include chimeric antibodies comprisingheavy chain CDR1, CDR2, and CDR3, and/or light chain CDR1, CDR2, andCDR3 of an antibody selected from antibody 11606; antibody 11613;antibody 11645; antibody 12191; antibody 12195; antibody 12220; antibody12228; antibody 12535; antibody 12536; antibody 12541; antibody 12543;antibody 12544; antibody 12545; antibody 12549; antibody 12550; antibody12553; antibody 12554; antibody 12562; antibody 12563; antibody 12564;antibody 12565; antibody 12571; antibody 12572; antibody 12576; antibody12583; antibody 12584; antibody 13396; antibody 13398; antibody 13399;antibody 13401; antibody 13402; antibody 13403; antibody 13404; antibody13405; antibody 13406; antibody 13407; antibody 13408; antibody 13409;antibody 11624; and antibody 12190, as disclosed herein. In someembodiments, the chimeric anti-PD-1 antibody comprises the variableregions described above and binds to PD-1. In some embodiments, thechimeric anti-PD-1 antibody comprises the variable regions describedabove, binds to PD-1 and inhibits binding of PD-1 to PD-L1 and/or PD-L2.In some embodiments, the anti-PD-1 antibody comprises the variableregions described above, binds to PD-1 and inhibits binding of PD-1 toPD-L1. In some embodiments, the anti-PD-1 antibody comprises thevariable regions described above, binds to PD-1 and inhibits binding ofPD-1 to PD-L1 and PD-L2. In some embodiments, the anti-PD-1 antibodycomprises the variable regions described above, binds to PD-1 andenhances an immune response in a subject, and/or increases activation ofT cells in a subject following administration of the antibody to thesubject. In some embodiments, administration of the anti-PD-1 antibodiesdescribed herein stimulates the activity of an immune cell, reduces thedownmodulation of an immune cell, or increases a T cell response in asubject.

In some embodiments, a chimeric anti-PD-1 antibody comprises a heavychain comprising a variable region sequence that is at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to asequence selected from SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68,76, 84, 92, 100, 108, 116, 124, 132, 140, 148, 156, 164, 172, 180, 188,196, 204, 212, 220, 228, 236, 244, 252, 260, 268, 276, 284, 292, 300,308, or 316, wherein the antibody binds PD-1. In some embodiments, achimeric anti-PD-1 antibody comprises a light chain comprising avariable region sequence that is at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to a sequence selected fromSEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112,120, 128, 136, 144, 152, 160, 168, 176, 184, 192, 200, 208, 216, 224,232, 240, 248, 256, 264, 272, 280, 288, 296, 304, 312 or 320, whereinthe antibody binds PD-1. In some embodiments, a chimeric anti-PD-1antibody comprises a heavy chain comprising a variable region sequencethat is at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical to a sequence selected from SEQ ID NOs: 4, 12, 20, 28, 36,44, 52, 60, 68, 76, 84, 92, 100, 108, 116, 124, 132, 140, 148, 156, 164,172, 180, 188, 196, 204, 212, 220, 228, 236, 244, 252, 260, 268, 276,284, 292, 300, 308, or 316; and a light chain comprising a variableregion sequence that is at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to a sequence selected from SEQ IDNOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128,136, 144, 152, 160, 168, 176, 184, 192, 200, 208, 216, 224, 232, 240,248, 256, 264, 272, 280, 288, 296, 304, 312 or 320; wherein the antibodybinds PD-1.

Exemplary chimeric anti-PD-1 antibodies also include chimeric antibodiesthat compete for binding to PD-1 with an antibody or fragment thereofdescribed herein. Thus, in some embodiments, a chimeric anti-PD-1antibody is provided that competes for binding to PD-1 with an antibodyselected from antibody 11606; antibody 11613; antibody 11645; antibody12191; antibody 12195; antibody 12220; antibody 12228; antibody 12535;antibody 12536; antibody 12541; antibody 12543; antibody 12544; antibody12545; antibody 12549; antibody 12550; antibody 12553; antibody 12554;antibody 12562; antibody 12563; antibody 12564; antibody 12565; antibody12571; antibody 12572; antibody 12576; antibody 12583; antibody 12584;antibody 13396; antibody 13398; antibody 13399; antibody 13401; antibody13402; antibody 13403; antibody 13404; antibody 13405; antibody 13406;antibody 13407; antibody 13408; antibody 13409; antibody 11624; andantibody 12190, or a fragment thereof. In some embodiments, the chimericanti-PD-1 antibody competes for binding to PD-1 with an antibodydescribed herein and inhibits binding of PD-1 to PD-L1 and/or PD-L2. Insome embodiments, the chimeric anti-PD-1 antibody competes for bindingto PD-1 with an antibody described herein and inhibits binding of PD-1to PD-L1. In some embodiments, the chimeric anti-PD-1 antibody competesfor binding to PD-1 with an antibody described herein and inhibitsbinding of PD-1 to PD-L1 and PD-L2. In some embodiments, the chimericanti-PD-1 antibody competes for binding to PD-1 with an antibodydescribed herein and enhances an immune response in a subject, and/orincreases activation of T cells in a subject following administration ofthe antibody to the subject.

In some embodiments, a chimeric antibody described herein comprises oneor more human constant regions. In some embodiments, the human heavychain constant region is of an isotype selected from IgA, IgG, and IgD.In some embodiments, the human light chain constant region is of anisotype selected from κ and λ. In some embodiments, a chimeric antibodydescribed herein comprises a human IgG constant region. In someembodiments, a chimeric antibody described herein comprises a human IgG4heavy chain constant region. In some embodiments, a chimeric antibodydescribed herein comprises a human IgG4 constant region and a human κlight chain.

As noted above, whether or not effector function is desirable may dependon the particular method of treatment intended for an antibody. Thus, insome embodiments, when effector function is desirable, a chimericanti-PD-1 antibody comprising a human IgG1 heavy chain constant regionor a human IgG3 heavy chain constant region is selected. In someembodiments, when effector function is not desirable, a chimericanti-PD-1 antibody comprising a human IgG4 or IgG2 heavy chain constantregion is selected.

Exemplary Humanized Antibodies

In some embodiments, humanized antibodies that bind PD-1 are provided.Humanized antibodies are useful as therapeutic molecules becausehumanized antibodies reduce or eliminate the human immune response ascompared to non-human antibodies, which can result in an immune responseto an antibody therapeutic (such as the human anti-mouse antibody (HAMA)response), and decreased effectiveness of the therapeutic.

In some embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which CDRs, (or portions thereof) are derivedfrom a non-human antibody, and FRs (or portions thereof) are derivedfrom human antibody sequences. A humanized antibody optionally will alsocomprise at least a portion of a human constant region. In someembodiments, some FR residues in a humanized antibody are substitutedwith corresponding residues from a non-human antibody (for example, theantibody from which the CDR residues are derived), for example, torestore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, forexample, in Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633,and are further described, for example, in Riechmann et al., (1988)Nature 332:323-329; Queen et al., (1989) Proc. Natl Acad. Sci. USA 86:10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and7,087,409; Kashmiri et al., (2005) Methods 36:25-34; Padlan, (1991) Mol.Immunol. 28:489-498 (describing “resurfacing”); Dall′Acqua et al.,(2005) Methods 36:43-60 (describing “FR shuffling”); and Osbourn et al.,(2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer,83:252-260 (describing the “guided selection” approach to FR shuffling).

Human framework regions that can be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, for example, Sims et al. (1993) J. Immunol. 151:2296);framework regions derived from the consensus sequence of humanantibodies of a particular subgroup of light or heavy chain variableregions (see, for example, Carter et al. (1992) Proc. Natl. Acad. Sci.USA, 89:4285; and Presta et al. (1993) J. Immunol, 151:2623); humanmature (somatically mutated) framework regions or human germlineframework regions (see, for example, Almagro and Fransson, (2008) Front.Biosci. 13:1619-1633); and framework regions derived from screening FRlibraries (see, for example, Baca et al., (1997) J. Biol. Chem. 272:10678-10684 and Rosok et al., (1996) J. Biol. Chem. 271: 22611-22618).

Exemplary humanized anti-PD-1 antibodies include antibodies that competefor binding to PD-1 with an antibody or fragment thereof describedherein. Thus, in some embodiments, a humanized anti-PD-1 antibody isprovided that competes for binding to PD-1 with an antibody or fragmentthereof selected from antibody 11606; antibody 11613; antibody 11645;antibody 12191; antibody 12195; antibody 12220; antibody 12228; antibody12535; antibody 12536; antibody 12541; antibody 12543; antibody 12544;antibody 12545; antibody 12549; antibody 12550; antibody 12553; antibody12554; antibody 12562; antibody 12563; antibody 12564; antibody 12565;antibody 12571; antibody 12572; antibody 12576; antibody 12583; antibody12584; antibody 13396; antibody 13398; antibody 13399; antibody 13401;antibody 13402; antibody 13403; antibody 13404; antibody 13405; antibody13406; antibody 13407; antibody 13408; antibody 13409; antibody 11624;and antibody 12190. In some embodiments, the humanized anti-PD-1antibody competes for binding to PD-1 with an antibody described hereinand inhibits binding of PD-1 to PD-L1 and/or PD-L2. In some embodiments,the humanized anti-PD-1 antibody competes for binding to PD-1 with anantibody described herein and inhibits binding of PD-1 to PD-L1. In someembodiments, the humanized anti-PD-1 antibody competes for binding toPD-1 with an antibody described herein and inhibits binding of PD-1 toPD-L1 and PD-L2. In some embodiments, the humanized anti-PD-1 antibodycompetes for binding to PD-1 with an antibody described herein andenhances an immune response in a subject, and/or increases activation ofT cells in a subject following administration of the antibody to thesubject.

Exemplary Human Antibodies

In some embodiments, an anti-PD-1 antibody provided herein is a humanantibody. Human antibodies can be produced using various techniquesknown in the art. Human antibodies are described generally in van Dijkand van de Winkel, (2001) Curr. Opin. Pharmacol. 5:368-374 and Lonberg,(2008) Curr. Opin. Immunol. 20:450-459. In some embodiments, the humanantibody is not a naturally occurring antibody. In some embodiments, thehuman antibody is a monoclonal antibody; thus, in some embodiments, eachof the human antibodies in a set can bind to the same epitope on theantigen.

Human antibodies can be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, (2005) Nat. Biotech. 23: 1117-1125. Seealso, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584 describingXENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HUMAB®technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology,and U.S. Patent Application Publication No. US 2007/0061900, describingVELOCIMOUSE® technology). Human variable regions from intact antibodiesgenerated by such animals may be further modified, for example, bycombining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, for example,Kozbor (1984) J. Immunol, 133: 3001; Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al, (1991) J. Immunol., 147:86). Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., (2006) Proc. Natl. Acad. Sci. USA,103:3557-3562. Additional methods include those described, for example,in U.S. Pat. No. 7,189,826 (describing production of monoclonal humanIgM antibodies from hybridoma cell lines) and Ni, (2006) XiandaiMianyixue, 26(4):265-268 (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, (2005) Histology and Histopathology, 20(3):927-937 (2005)and Vollmers and Brandlein, (2005) Methods and Findings in Experimentaland Clinical Pharmacology, 27(3): 185-191.

Human antibodies can also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

Antibodies may be isolated by screening combinatorial libraries forantibodies with the desired activity or activities. For example, avariety of methods are known in the art for generating phage displaylibraries and screening such libraries for antibodies possessing thedesired binding characteristics. Such methods are reviewed, for example,in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brienet al., ed., Human Press, Totowa, N.J., 2001) and further described, forexample, in the McCafferty et al, (1990) Nature 348:552-554; Clackson etal, (1991) Nature 352: 624-628; Marks et al, (1992) J. Mol. Biol 222:581-597; Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et al, (2004)J. Mol. Biol. 338(2): 299-310; Lee et al., (2004) J. Mol. Biol. 340(5):1073-1093; Fellouse, (2004) Proc. Natl. Acad. Sci. USA 101(34):12467-12472; and Lee et al, (2004) J. Immunol. Methods 284(1-2): 119-132and PCT publication WO 99/10494.

In certain phage display methods, repertoires of V_(H) and V_(L) genesare separately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., (1994) Ann. Rev.Immunol., 12:433-455. Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (for example, from human) to provide asingle source of antibodies to a wide range of non-self and alsoself-antigens without any immunization as described by Griffiths et al.,(1993) EMBO J 12:725-734. Finally, naive libraries can also be madesynthetically by cloning unrearranged V-gene segments from stem cells,and using PCR primers containing random sequence to encode the highlyvariable CDR3 regions and to accomplish rearrangement in vitro, asdescribed by Hoogenboom and Winter (1992), J. Mol. Biol, 227:381-388.Patent publications describing human antibody phage libraries include,for example: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

In some embodiments, a human anti-PD-1 antibody binds to a polypeptidehaving the sequence of SEQ ID NO: 1, 2, 3, 382, 383, or 384. In someembodiments, the human anti-PD-1 antibody binds to PD-1 and inhibitsbinding of PD-1 to PD-L1 and/or PD-L2. In some embodiments, the humananti-PD-1 antibody binds to PD-1 and inhibits binding of PD-1 to PD-L1.In some embodiments, the human anti-PD-1 antibody binds to PD-1 andinhibits binding of PD-1 to PD-L1 and PD-L2. In some embodiments, thehuman anti-PD-1 antibody binds to PD-1 and enhances an immune responsein a subject, and/or increases activation of T cells in a subjectfollowing administration of the antibody to the subject.

Exemplary human anti-PD-1 antibodies also include antibodies thatcompete for binding to PD-1 with a human antibody or fragment thereofdescribed herein. Thus, in some embodiments, a human anti-PD-1 antibodyis provided that competes for binding to PD-1 with an antibody orfragment thereof selected from antibody 11606; antibody 11613; antibody11645; antibody 12191; antibody 12195; antibody 12220; antibody 12228;antibody 12535; antibody 12536; antibody 12541; antibody 12543; antibody12544; antibody 12545; antibody 12549; antibody 12550; antibody 12553;antibody 12554; antibody 12562; antibody 12563; antibody 12564; antibody12565; antibody 12571; antibody 12572; antibody 12576; antibody 12583;antibody 12584; antibody 13396; antibody 13398; antibody 13399; antibody13401; antibody 13402; antibody 13403; antibody 13404; antibody 13405;antibody 13406; antibody 13407; antibody 13408; antibody 13409; antibody11624; and antibody 12190. In some embodiments, the human anti-PD-1antibody competes for binding to PD-1 with an antibody described hereinand inhibits binding of PD-1 to PD-L1 and/or PD-L2. In some embodiments,the human anti-PD-1 antibody competes for binding to PD-1 with anantibody described herein and inhibits binding of PD-1 to PD-L1. In someembodiments, the human anti-PD-1 antibody competes for binding to PD-1with an antibody described herein and inhibits binding of PD-1 to PD-L1and PD-L2. In some embodiments, the human anti-PD-1 antibody competesfor binding to PD-1 with an antibody described herein and enhances animmune response in a subject, and/or increases activation of T cells ina subject following administration of the antibody to the subject.

In some embodiments, a chimeric human anti-PD-1 antibody is provided,where the antibody comprises the variable region from a human antibodythat binds PD-1 and the constant region from a different human antibody.In some embodiments, a chimeric human anti-PD-1 antibody, where theantibody comprises the CDRs from a human antibody that binds PD-1 and aframework from a different human antibody is provided. In someembodiments, the antibody is not a naturally occurring human antibody.

In some embodiments, a human anti-PD-1 antibody comprises one or morehuman constant regions. In some embodiments, the human heavy chainconstant region is of an isotype selected from IgA, IgG, and IgD. Insome embodiments, the human light chain constant region is of an isotypeselected from κ and λ. In some embodiments, a human antibody describedherein comprises a human IgG constant region. In some embodiments, ahuman antibody described herein comprises a human IgG4 heavy chainconstant region. In some embodiments, a human antibody described hereincomprises a human IgG4 constant region and a human κ light chain.

In some embodiments, when effector function is desirable, a humananti-PD-1 antibody comprising a human IgG1 heavy chain constant regionor a human IgG3 heavy chain constant region is selected. In someembodiments, when effector function is not desirable, a human anti-PD-1antibody comprising a human IgG4 or IgG2 heavy chain constant region isselected.

As noted herein, the term “human antibody” denotes the genus of possiblesequences for the antibody construct, rather than a source of theantibody.

Exemplary Antibody Constant Regions

In some embodiments, an antibody described herein comprises one or morehuman constant regions. In some embodiments, the human heavy chainconstant region is of an isotype selected from IgA, IgG, and IgD. Insome embodiments, an antibody described herein comprises a human IgGconstant region. In some embodiments, when effector function isdesirable, an anti-PD-1 antibody comprising a human IgG1 heavy chainconstant region or a human IgG3 heavy chain constant region is selected.In some embodiments, when effector function is not desirable, ananti-PD-1 antibody comprising a human IgG4 or IgG2 heavy chain constantregion is selected. In some embodiments, the human light chain constantregion is of an isotype selected from κ and λ. In some embodiments, anantibody described herein comprises a human IgG4 heavy chain constantregion. In some embodiments, an antibody described herein comprises ahuman IgG4 constant region and a human κ light chain.

Throughout the present specification and claims unless explicitly statedor known to one skilled in the art, the numbering of the residues in animmunoglobulin heavy chain is that of the EU index as in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991), expresslyincorporated herein by reference. The “EU index as in Kabat” refers tothe residue numbering of the human IgG1 EU antibody.

As noted above, whether or not effector function is desirable may dependon the particular method of treatment intended for an antibody. Thus, insome embodiments, when effector function is desirable, an anti-PD-1antibody comprising a human IgG1 heavy chain constant region or a humanIgG3 heavy chain constant region is selected. In some embodiments, wheneffector function is not desirable, an anti-PD-1 antibody comprising ahuman IgG4 or IgG2 heavy chain constant region is selected.

In some embodiments, an antibody comprises a variant Fc region has atleast one amino acid substitution compared to the Fc region of awild-type IgG or a wild-type antibody. In some embodiments, the variantFc region has two or more amino acid substitutions in the Fc region ofthe wild-type antibody. In some embodiments, the variant Fc region hasthree or more amino acid substitutions in the Fc region of the wild-typeantibody. In some embodiments, the variant Fc region has at least one,two or three or more Fc region amino acid substitutions describedherein. In some embodiments, the variant Fc region herein will possessat least about 80% homology with a native sequence Fc region and/or withan Fc region of a parent polypeptide. In some embodiments, the variantFc region herein will possess at least about 90% homology with a nativesequence Fc region and/or with an Fc region of a parent polypeptide. Insome embodiments, the variant Fc region herein will possess at leastabout 95% homology with a native sequence Fc region and/or with an Fcregion of a parent polypeptide. In some embodiments, a heavy chainconstant region lacks the C-terminal lysine (K) residue. In some suchembodiments, the heavy chain or heavy chain constant region may bereferred to as “desK.” In some embodiments, the heavy chain constantregion lacking the C-terminal lysine is an IgG, such as an IgG1, IgG2,IgG3, or IgG4.

In some embodiments, an antibody provided herein is altered to increaseor decrease the extent to which the antibody is glycosylated. Additionor deletion of glycosylation sites to an antibody may be convenientlyaccomplished by altering the amino acid sequence such that one or moreglycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, for example, mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody may be made in order to create antibodyvariants with certain improved properties.

In some embodiments, antibody variants are provided having acarbohydrate structure that lacks fucose attached (directly orindirectly) to an Fc region. For example, the amount of fucose in suchantibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from20% to 40%. The amount of fucose is determined by calculating theaverage amount of fucose within the sugar chain at Asn297, relative tothe sum of all glycostructures attached to Asn 297 (for example,complex, hybrid and high mannose structures) as measured by MALDI-TOFmass spectrometry, as described in WO 2008/077546, for example. Asn297refers to the asparagine residue located at about position 297 in the Fcregion (EU numbering of Fc region residues); however, Asn297 may also belocated about +3 amino acids upstream or downstream of position 297,that is, between positions 294 and 300, due to minor sequence variationsin antibodies. Such fucosylation variants may have improved ADCCfunction. See, for example, US Patent Publication Nos. US 2003/0157108(Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples ofpublications related to “defucosylated” or “fucose-deficient” antibodyvariants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742;WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004);Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of celllines capable of producing defucosylated antibodies include Lec13 CHOcells deficient in protein fucosylation (Ripka et al. Arch. Biochem.Biophys. 249:533-545 (1986); US Patent Application No. US 2003/0157108A1, Presta, L; and WO 2004/056312 A1, Adams et al., especially atExample 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, forexample, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y.et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibody variants are further provided with bisected oligosaccharides,for example, in which a biantennary oligosaccharide attached to the Fcregion of the antibody is bisected by GlcNAc. Such antibody variants mayhave reduced fucosylation and/or improved ADCC function. Examples ofsuch antibody variants are described, for example, in WO 2003/011878(Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and US2005/0123546 (Umana et al.). Antibody variants with at least onegalactose residue in the oligosaccharide attached to the Fc region arealso provided. Such antibody variants may have improved CDC function.Such antibody variants are described, for example, in WO 1997/30087(Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

Antibody variants are also provided with amino-terminal leaderextensions. For example, one or more amino acid residues of theamino-terminal leader sequence are present at the amino-terminus of anyone or more heavy or light chains of an antibody. An exemplaryamino-terminal leader extension comprises or consists of three aminoacid residues, VHS, present on one or both light chains of an antibodyvariant.

The in vivo or serum half-life of human FcRn high affinity bindingpolypeptides can be assayed, for example, in transgenic mice, in humans,or in non-human primates to which the polypeptides with a variant Fcregion are administered. See also, for example, Petkova et al.International Immunology 18(12):1759-1769 (2006).

In some embodiments, the antibody variant mediates ADCC in the presenceof human effector cells more effectively than a parent antibody. In someembodiments, the antibody variant is substantially more effective atmediating ADCC in vitro, when the amounts of polypeptide variant andparent antibody used in the assay are essentially the same. In someembodiments, the antibody variant is substantially more effective atmediating ADCC in vivo, when the amounts of polypeptide variant andparent antibody used in the assay are essentially the same. Generally,such variants will be identified using the in vitro ADCC assay as hereindisclosed, but other assays or methods for determining ADCC activity,for example in an animal model etc., are contemplated.

Exemplary Antibody Conjugates

In some embodiments, an anti-PD-1 antibody is conjugated to anothermolecule. In some embodiments, the additional molecule can be adetectable marker, such as a label. In some embodiments, the additionalmolecule can be a therapeutic molecule, such as a cytotoxic agent. Insome embodiments, a label and/or a cytotoxic agent can be conjugated tothe antibody. As used herein, a label is a moiety that facilitatesdetection of the antibody and/or facilitates detection of a molecule towhich the antibody binds. Nonlimiting exemplary labels include, but arenot limited to, radioisotopes, fluorescent groups, enzymatic groups,chemiluminescent groups, biotin, epitope tags, metal-binding tags, etc.One skilled in the art can select a suitable label according to thespecific application.

As used herein, a cytotoxic agent is a moiety that reduces theproliferative capacity of one or more cells. A cell has reducedproliferative capacity when the cell becomes less able to proliferate,for example, because the cell undergoes apoptosis or otherwise dies, thecell fails to proceed through the cell cycle and/or fails to divide, thecell differentiates, etc. Nonlimiting exemplary cytotoxic agentsinclude, but are not limited to, radioisotopes, toxins, andchemotherapeutic agents. One skilled in the art can select a suitablecytotoxic according to the intended application. In some embodiments,the cytotoxic agent is at least one of an anti-metabolite, an alkylatingagent, an antibiotic, a growth factor, a cytokine, an anti-angiogenicagent, an anti-mitotic agent, an anthracycline, toxin, or an apoptoticagent

In some embodiments, a label and/or a cytotoxic agent is conjugated toan antibody using chemical methods in vitro. Nonlimiting exemplarychemical methods of conjugation are known in the art, and includeservices, methods and/or reagents commercially available from, forexample, Thermo Scientific Life Science Research Produces (formerlyPierce; Rockford, Ill.), Prozyme (Hayward, Calif.), SACRI AntibodyServices (Calgary, Canada), AbD Serotec (Raleigh, N.C.), etc. In someembodiments, when a label and/or cytotoxic agent is a polypeptide, thelabel and/or cytotoxic agent can be expressed from the same expressionvector with at least one antibody chain to produce a polypeptidecomprising the label and/or cytotoxic agent fused to an antibody chain.One skilled in the art can select a suitable method for conjugating alabel and/or cytotoxic agent to an antibody according to the intendedapplication.

In some embodiments, conjugation can be covalent. In some embodiments,conjugation can be non-covalent. In some embodiments, conjugation can bevia a specific binding interaction, for example, through the binding ofa secondary antibody.

Exemplary Leader Sequences

In order for some secreted proteins to express and secrete in largequantities, a leader sequence from a heterologous protein may bedesirable. In some embodiments, employing heterologous leader sequencescan be advantageous in that a resulting mature polypeptide can remainunaltered as the leader sequence is removed in the ER during thesecretion process. The addition of a heterologous leader sequence can beuseful to express and secrete some proteins.

Certain exemplary leader sequence sequences are described, for example,in the online Leader sequence Database maintained by the Department ofBiochemistry, National University of Singapore. See Choo et al., BMCBioinformatics, 6: 249 (2005); and PCT Publication No. WO 2006/081430.

III. Antibody Expression and Production Nucleic Acid Molecules EncodingAnti-PD-1 Antibodies

Nucleic acid molecules comprising polynucleotides that encode one ormore chains of an anti-PD-1 antibody are provided herein. In someembodiments, a nucleic acid molecule comprises a polynucleotide thatencodes a heavy chain or a light chain of an anti-PD-1 antibody. In someembodiments, a nucleic acid molecule comprises both a polynucleotidethat encodes a heavy chain and a polynucleotide that encodes a lightchain, of an anti-PD-1 antibody. In some embodiments, a first nucleicacid molecule comprises a first polynucleotide that encodes a heavychain and a second nucleic acid molecule comprises a secondpolynucleotide that encodes a light chain.

In some embodiments, the heavy chain and the light chain are expressedfrom one nucleic acid molecule, or from two separate nucleic acidmolecules, as two separate polypeptides. In some embodiments, such aswhen an antibody is an scFv, a single polynucleotide encodes a singlepolypeptide comprising both a heavy chain and a light chain linkedtogether.

In some embodiments, a polynucleotide encoding a heavy chain or lightchain of an anti-PD-1 antibody comprises a nucleotide sequence thatencodes at least one of the CDRs provided herein. In some embodiments, apolynucleotide encoding a heavy chain or light chain of an anti-PD-1antibody comprises a nucleotide sequence that encodes at least 3 of theCDRs provided herein. In some embodiments, a polynucleotide encoding aheavy chain or light chain of an anti-PD-1 antibody comprises anucleotide sequence that encodes at least 6 of the CDRs provided herein.In some embodiments, a polynucleotide encoding a heavy chain or lightchain of an anti-PD-1 antibody comprises a nucleotide sequence thatencodes a leader sequence, which, when translated, is located at the Nterminus of the heavy chain or light chain. As discussed above, theleader sequence may be the native heavy or light chain leader sequence,or may be another heterologous leader sequence.

In some embodiments, the nucleic acid is one that encodes for any of theamino acid sequences for the antibodies in the Sequence Table herein. Insome embodiments, the nucleic acid is one that is at least 80% identicalto a nucleic acid encoding any of the amino acid sequences for theantibodies in the Sequence Table herein, for example, at least 80, 85,90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical. In someembodiments, the nucleic acid is one that hybridizes to any one or moreof the nucleic acid sequences provided herein. In some of theembodiments, the hybridization is under moderate conditions. In someembodiments, the hybridization is under highly stringent conditions,such as: at least about 6×SSC and 1% SDS at 65° C., with a first washfor 10 minutes at about 42° C. with about 20% (v/v) formamide in0.1×SSC, and with a subsequent wash with 0.2×SSC and 0.1% SDS at 65° C.

Nucleic acid molecules can be constructed using recombinant DNAtechniques conventional in the art. In some embodiments, a nucleic acidmolecule is an expression vector that is suitable for expression in aselected host cell.

Vectors comprising polynucleotides that encode anti-PD-1 heavy chainsand/or anti-PD-1 light chains are provided. Vectors comprisingpolynucleotides that encode anti-PD-1 heavy chains and/or anti-PD-1light chains are also provided. Such vectors include, but are notlimited to, DNA vectors, phage vectors, viral vectors, retroviralvectors, etc. In some embodiments, a vector comprises a firstpolynucleotide sequence encoding a heavy chain and a secondpolynucleotide sequence encoding a light chain. In some embodiments, theheavy chain and light chain are expressed from the vector as twoseparate polypeptides. In some embodiments, the heavy chain and lightchain are expressed as part of a single polypeptide, such as, forexample, when the antibody is an scFv.

In some embodiments, a first vector comprises a polynucleotide thatencodes a heavy chain and a second vector comprises a polynucleotidethat encodes a light chain. In some embodiments, the first vector andsecond vector are transfected into host cells in similar amounts (suchas similar molar amounts or similar mass amounts). In some embodiments,a mole- or mass-ratio of between 5:1 and 1:5 of the first vector and thesecond vector is transfected into host cells. In some embodiments, amass ratio of between 1:1 and 1:5 for the vector encoding the heavychain and the vector encoding the light chain is used. In someembodiments, a mass ratio of 1:2 for the vector encoding the heavy chainand the vector encoding the light chain is used.

In some embodiments, a vector is selected that is optimized forexpression of polypeptides in CHO or CHO-derived cells, or in NSO cells.Exemplary such vectors are described, for example, in Running Deer etal., Biotechnol. Prog. 20:880-889 (2004).

Host Cells

In some embodiments, anti-PD-1 antibody heavy chains and/or anti-PD-1antibody light chains may be expressed in prokaryotic cells, such asbacterial cells; or in eukaryotic cells, such as fungal cells (such asyeast), plant cells, insect cells, and mammalian cells. Such expressionmay be carried out, for example, according to procedures known in theart. Exemplary eukaryotic cells that may be used to express polypeptidesinclude, but are not limited to, COS cells, including COS 7 cells; 293cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lec13CHO cells, and FUT8 CHO cells; PER.C6® cells (Crucell); and NSO cells.In some embodiments, anti-PD-1 antibody heavy chains and/or anti-PD-1antibody light chains may be expressed in yeast. See, for example, U.S.Publication No. US 2006/0270045 A1. In some embodiments, a particulareukaryotic host cell is selected based on its ability to make desiredpost-translational modifications to the anti-PD-1 antibody heavy chainsand/or anti-PD-1 antibody light chains. For example, in someembodiments, CHO cells produce polypeptides that have a higher level ofsialylation than the same polypeptide produced in 293 cells.

Introduction of one or more nucleic acids into a desired host cell maybe accomplished by any method, including but not limited to, calciumphosphate transfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection,etc. Nonlimiting exemplary methods are described, for example, inSambrook et al., Molecular Cloning, A Laboratory Manual, 3^(rd) ed. ColdSpring Harbor Laboratory Press (2001). Nucleic acids may be transientlyor stably transfected in the desired host cells, according to anysuitable method.

Host cells comprising any of the polynucleotides or vectors describedherein are also provided. In some embodiments, a host cell comprising ananti-PD-1 antibody is provided. Any host cells capable ofover-expressing heterologous DNAs can be used for the purpose ofisolating the genes encoding the antibody, polypeptide or protein ofinterest. Non-limiting examples of mammalian host cells include but notlimited to COS, HeLa, and CHO cells. See also PCT Publication No. WO87/04462. Suitable non-mammalian host cells include prokaryotes (such asE. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; orK. lactis).

Purification of Antibodies

Anti-PD-1 antibodies can be purified by any suitable method. Suchmethods include, but are not limited to, the use of affinity matrices orhydrophobic interaction chromatography. Suitable affinity ligandsinclude the ROR1 ECD and ligands that bind antibody constant regions.For example, a Protein A, Protein G, Protein A/G, or an antibodyaffinity column may be used to bind the constant region and to purify ananti-PD-1 antibody. Hydrophobic interactive chromatography, for example,a butyl or phenyl column, may also suitable for purifying somepolypeptides such as antibodies. Ion exchange chromatography (forexample anion exchange chromatography and/or cation exchangechromatography) may also suitable for purifying some polypeptides suchas antibodies. Mixed-mode chromatography (for example reversedphase/anion exchange, reversed phase/cation exchange, hydrophilicinteraction/anion exchange, hydrophilic interaction/cation exchange,etc.) may also suitable for purifying some polypeptides such asantibodies. Many methods of purifying polypeptides are known in the art.

Cell-Free Production of Antibodies

In some embodiments, an anti-PD-1 antibody is produced in a cell-freesystem. Nonlimiting exemplary cell-free systems are described, forexample, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009);Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol.Adv. 21: 695-713 (2003).

Compositions

In some embodiments, antibodies prepared by the methods described aboveare provided. In some embodiments, the antibody is prepared in a hostcell. In some embodiments, the antibody is prepared in a cell-freesystem. In some embodiments, the antibody is purified. In someembodiments, the antibody prepared in a host cell or a cell-free systemis a chimeric antibody. In some embodiments, the antibody prepared in ahost cell or a cell-free system is a humanized antibody. In someembodiments, the antibody prepared in a host cell or a cell-free systemis a human antibody. In some embodiments, a cell culture mediacomprising an anti-PD-1 antibody is provided. In some embodiments, ahost cell culture fluid comprising an anti-PD-1 antibody is provided.

In some embodiments, compositions comprising antibodies prepared by themethods described above are provided. In some embodiments, thecomposition comprises an antibody prepared in a host cell. In someembodiments, the composition comprises an antibody prepared in acell-free system. In some embodiments, the composition comprises apurified antibody. In some embodiments, the composition comprises achimeric antibody prepared in a host cell or a cell-free system. In someembodiments, the composition comprises a humanized antibody prepared ina host cell or a cell-free system. In some embodiments, the compositioncomprises a human antibody prepared in a host cell or a cell-freesystem.

In some embodiments, a composition comprising anti-PD-1 antibody at aconcentration of more than about any one of 10 mg/mL, 20 mg/mL, 30mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100mg/mL, 125 mg/mL, 150 mg/mL, 175 mg/mL, 200 mg/mL, 225 mg/mL, or 250mg/mL is provided. In some embodiments, the composition comprises achimeric antibody prepared in a host cell or a cell-free system. In someembodiments, the composition comprises a humanized antibody prepared ina host cell or a cell-free system. In some embodiments, the compositioncomprises a human antibody prepared in a host cell or a cell-freesystem.

IV. Therapeutic Compositions and Methods Methods of Treating DiseasesUsing Anti-PD-1 Antibodies

Antibodies and compositions comprising antibodies are provided for usein methods of treatment for humans or animals. Methods of treatingdisease comprising administering anti-PD-1 antibodies are also provided.Nonlimiting exemplary diseases that can be treated with anti-PD-1antibodies include, but are not limited to, cancer.

In some embodiments, a method of treating cancer is provided, whereincells within a sample of the tumor express PD-L1. In some suchembodiments, the tumor may be considered to be PD-L1-positive, or toexpress PD-L1. Expression of PD-L1 may be determined by IHC, e.g., asdiscussed herein. In some embodiments, a tumor is considered to expressPD-L1 when a sample from the tumor shows 1+, 2+, or 3+ staining of PD-L1by IHC. In some embodiments, the sample from the tumor shows 2+ or 3+staining of PD-L1 by IHC. In some embodiments, a tumor sample from asubject is analyzed for PD-L1 expression and the subject is selected fortreatment with an antibody described herein if the tumor sample showsPD-L1 expression. In some embodiments, the subject is selected if thetumor sample shows elevated expression of PD-L1.

In some embodiments, a subject is selected for treatment with ananti-PD-1 antibody provided herein if the subject's tumor isPD-L1^(HIGH). In some embodiments, a subject is selected for treatmentwith an anti-PD-1 antibody provided herein if the subject's tumor isPD-L1^(LOW). In some embodiments, a subject is selected for treatmentwith an anti-PD-1 antibody provided herein if the subject's tumor isPD-1^(HIGH)/PD-L1^(LOW). In some embodiments, a subject is selected fortreatment with an anti-PD-1 antibody provided herein if the subject'stumor is PD-1^(HIGH)/PD-L1^(HIGH).

The anti-PD-1 antibody can be administered as needed to subjects.Determination of the frequency of administration can be made by personsskilled in the art, such as an attending physician based onconsiderations of the condition being treated, age of the subject beingtreated, severity of the condition being treated, general state ofhealth of the subject being treated and the like. In some embodiments,an effective dose of an anti-PD-1 antibody is administered to a subjectone or more times. In some embodiments, an effective dose of ananti-PD-1 antibody is administered to the subject once a month, lessthan once a month, such as, for example, every two months or every threemonths. In some embodiments, an effective dose of an anti-PD-1 antibodyis administered less than once a month, such as, for example, once everythree weeks, once every two weeks, or once every week. An effective doseof an anti-PD-1 antibody is administered to the subject at least once.In some embodiments, the effective dose of an anti-PD-1 antibody may beadministered multiple times, including for periods of at least a month,at least six months, or at least a year.

In some embodiments, pharmaceutical compositions are administered in anamount effective for treatment of (including prophylaxis of) cancer. Thetherapeutically effective amount is typically dependent on the weight ofthe subject being treated, his or her physical or health condition, theextensiveness of the condition to be treated, or the age of the subjectbeing treated. In general, anti-PD-1 antibodies may be administered inan amount in the range of about 10 μg/kg body weight to about 100 mg/kgbody weight per dose. In some embodiments, anti-PD-1 antibodies may beadministered in an amount in the range of about 50 μg/kg body weight toabout 5 mg/kg body weight per dose. In some embodiments, anti-PD-1antibodies may be administered in an amount in the range of about 100μg/kg body weight to about 10 mg/kg body weight per dose. In someembodiments, anti-PD-1 antibodies may be administered in an amount inthe range of about 100 μg/kg body weight to about 20 mg/kg body weightper dose. In some embodiments, anti-PD-1 antibodies may be administeredin an amount in the range of about 0.5 mg/kg body weight to about 20mg/kg body weight per dose.

Pharmaceutical compositions are administered in an amount effective forenhancing an immune response and/or increasing T cell activation in asubject.

The therapeutically effective amount is typically dependent on theweight of the subject being treated, his or her physical or healthcondition, the extensiveness of the condition to be treated, or the ageof the subject being treated. In general, anti-PD-1 antibodies may beadministered in an amount in the range of about 10 μg/kg body weight toabout 100 mg/kg body weight per dose. In some embodiments, anti-PD-1antibodies may be administered in an amount in the range of about 50μg/kg body weight to about 5 mg/kg body weight per dose. In someembodiments, anti-PD-1 antibodies may be administered in an amount inthe range of about 100 g/kg body weight to about 10 mg/kg body weightper dose. In some embodiments, anti-PD-1 antibodies may be administeredin an amount in the range of about 100 μg/kg body weight to about 20mg/kg body weight per dose. In some embodiments, anti-PD-1 antibodiesmay be administered in an amount in the range of about 0.5 mg/kg bodyweight to about 20 mg/kg body weight per dose.

Pharmaceutical Compositions

In some embodiments, compositions comprising anti-PD-1 antibodies areprovided in formulations with a wide variety of pharmaceuticallyacceptable carriers (see, for example, Gennaro, Remington: The Scienceand Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus,20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and DrugDelivery Systems, 7^(th) ed., Lippencott Williams and Wilkins (2004);Kibbe et al., Handbook of Pharmaceutical Excipients, 3^(rd) ed.,Pharmaceutical Press (2000)). Various pharmaceutically acceptablecarriers, which include vehicles, adjuvants, and diluents, areavailable. Moreover, various pharmaceutically acceptable auxiliarysubstances, such as pH adjusting and buffering agents, tonicityadjusting agents, stabilizers, wetting agents and the like, are alsoavailable. Non-limiting exemplary carriers include saline, bufferedsaline, dextrose, water, glycerol, ethanol, and combinations thereof.

In some embodiments, a pharmaceutical composition comprising ananti-PD-1 antibody is provided. In some embodiments, the pharmaceuticalcomposition comprises a chimeric antibody. In some embodiments, thepharmaceutical composition comprises a humanized antibody. In someembodiments, the pharmaceutical composition comprises an antibodyprepared in a host cell or cell-free system as described herein. In someembodiments, the pharmaceutical composition comprises pharmaceuticallyacceptable carrier.

In some embodiments, pharmaceutical compositions are administered in anamount effective for treatment of (including prophylaxis of) cancer. Thetherapeutically effective amount is typically dependent on the weight ofthe subject being treated, his or her physical or health condition, theextensiveness of the condition to be treated, or the age of the subjectbeing treated. In general, anti-PD-1 antibodies may be administered inan amount in the range of about 0.05 mg/kg body weight to about 100mg/kg body weight per dose. In some embodiments, anti-PD-1 antibodiesmay be administered in an amount in the range of about 10 g/kg bodyweight to about 100 mg/kg body weight per dose. In some embodiments,anti-PD-1 antibodies may be administered in an amount in the range ofabout 50 μg/kg body weight to about 5 mg/kg body weight per dose. Insome embodiments, anti-PD-1 antibodies may be administered in an amountin the range of about 100 μg/kg body weight to about 10 mg/kg bodyweight per dose. In some embodiments, anti-PD-1 antibodies may beadministered in an amount in the range of about 100 μg/kg body weight toabout 20 mg/kg body weight per dose. In some embodiments, anti-PD-1antibodies may be administered in an amount in the range of about 0.5mg/kg body weight to about 20 mg/kg body weight per dose. In someembodiments, anti-PD-1 antibodies may be administered in an amount inthe range of about 0.5 mg/kg body weight to about 10 mg/kg body weightper dose. In some embodiments, anti-PD-1 antibodies may be administeredin an amount in the range of about 0.05 mg/kg body weight to about 20mg/kg body weight per dose. In some embodiments, anti-PD-1 antibodiesmay be administered in an amount in the range of about 0.05 mg/kg bodyweight to about 10 mg/kg body weight per dose. In some embodiments,anti-PD-1 antibodies may be administered in an amount in the range ofabout 5 mg/kg body weight or lower, for example less than 4, less than3, less than 2, or less than 1 mg/kg of the antibody.

In some embodiments, anti-PD-1 antibodies can be present in an amount inthe range of about 50 μg/kg body weight to about 5 mg/kg body weight perdose. For example, in some embodiments, a dose for a 20 kg person can bewithin a range of about 1 mg to about 100 mg. In some embodiments, thedose can be within a range of 2 mg to 200 mg of the anti-PD-1 antibody.In some embodiments, the dose can be within a range of 10 mg to 400 mgof the anti-PD-1 antibody.

Routes of Administration

In some embodiments, anti-PD-1 antibodies can be administered in vivo byvarious routes, including, but not limited to, intravenous,intra-arterial, parenteral, intratumoral, intraperitoneal orsubcutaneous. The appropriate formulation and route of administrationmay be selected according to the intended application.

Combination Therapy

Anti-PD-1 antibodies can be administered alone or with other modes oftreatment. They can be provided before, substantially contemporaneouswith, and/or after other modes of treatment, for example, surgery,chemotherapy, radiation therapy, or the administration of a biologic,such as another therapeutic antibody. In some embodiments, an anti-PD-1antibody is administered in conjunction with another anti-cancer agent.

In some embodiments, the anti-PD-1 antibody is given concurrently with asecond therapeutic agent. For example, the two or more therapeuticagents are administered with a time separation of no more than about 60minutes, such as no more than about any of 30, 15, 10, 5, or 1 minutes.In some embodiments, the anti-PD-1 antibody is administered sequentiallywith a second therapeutic agent. For example, administration of the twoor more therapeutic agents are administered with a time separation ofmore than about 15 minutes, such as about any of 20, 30, 40, 50, or 60minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month, or longer.

In some embodiments, the anti-PD-1 antibody is administered with asecond therapeutic method for treatment. Thus, the administration of anantibody provided herein can be in combination with another system oftreatment.

In some embodiments, an anti-PD-1 antibody provided herein isadministered with an anti-ICOS therapy. In some embodiments, ananti-PD-1 antibody provided herein is administered with an antibody thatbinds Inducible T-Cell Costimulator (ICOS). In some embodiments, theanti-PD-1 antibody provided herein is administered with an isolatedantibody that binds ICOS, wherein the anti-ICOS antibody is an agonistof CD4+ T cells (such as CD4+ T effector (Teff) cells). In someembodiments, the antibody that binds ICOS is an agonist of CD4+ T cells(such as CD4+ Teff cells) and depletes T regulatory (Treg) cells. Insome embodiments, the antibody that binds ICOS depletes Treg cells, butdoes not deplete Teff cells. In some embodiments, the antibody thatbinds ICOS induces pAKT signaling on CD4+ T cells. In some embodiments,the isolated antibody that binds ICOS induces pAKT signaling on CD4+ Tcells and depletes Treg cells. In some embodiments, the isolatedantibody that binds ICOS comprises:

-   -   i) (a) HCDR1 comprising the amino acid sequence of SEQ ID NO:        326; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO:        327; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO:        328; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO:        329; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO:        330; and (f) LCDR3 comprising the amino acid sequence of SEQ ID        NO: 331; or    -   ii) (a) HCDR1 comprising the amino acid sequence of SEQ ID NO:        334; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO:        335; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO:        336; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO:        337; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO:        338; and (f) LCDR3 comprising the amino acid sequence of SEQ ID        NO: 339; or    -   iii) (a) HCDR1 comprising the amino acid sequence of SEQ ID NO:        342; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO:        343; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO:        344; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO:        345; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO:        346; and (f) LCDR3 comprising the amino acid sequence of SEQ ID        NO: 347; or    -   iv) (a) HCDR1 comprising the amino acid sequence of SEQ ID NO:        350; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO:        351; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO:        352; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO:        353 (e) LCDR2 comprising the amino acid sequence of SEQ ID NO:        354; and (f) LCDR3 comprising the amino acid sequence of SEQ ID        NO: 355; or    -   v) (a) HCDR1 comprising the amino acid sequence of SEQ ID NO:        358; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO:        359; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO:        360; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO:        361; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO:        362; and (f) LCDR3 comprising the amino acid sequence of SEQ ID        NO: 363; or    -   vi) (a) HCDR1 comprising the amino acid sequence of SEQ ID NO:        366; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO:        367; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO:        368; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO:        369; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO:        370; and (f) LCDR3 comprising the amino acid sequence of SEQ ID        NO: 371; or    -   vii)(a) HCDR1 comprising the amino acid sequence of SEQ ID NO:        374; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO:        375; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO:        376; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO:        377; (e) LCDR2 comprising the amino acid sequence of SEQ ID NO:        378; and (f) LCDR3 comprising the amino acid sequence of SEQ ID        NO: 379.

In some embodiments, an antibody that binds to ICOS is provided, whereinthe antibody comprises a heavy chain variable region (V_(H)) and a lightchain variable region (V_(L)), wherein:

-   -   i) the V_(H) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,        98%, 99%, or 100% identical to the amino acid sequence of SEQ ID        NO: 324 and the V_(L) is at least 90%, 91%, 92%, 93%, 94%, 95%,        96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence        of SEQ ID NO: 325; or    -   ii) the V_(H) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, 99%, or 100% identical to the amino acid sequence of        SEQ ID NO: 332 and the V_(L) is at least 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino        acid sequence of SEQ ID NO: 333; or    -   iii) the V_(H) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, 99%, or 100% identical to the amino acid sequence of        SEQ ID NO: 340 and the V_(L) is at least 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino        acid sequence of SEQ ID NO: 341; or    -   iv) the V_(H) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, 99%, or 100% identical to the amino acid sequence of        SEQ ID NO: 348 and the V_(L) is at least 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino        acid sequence of SEQ ID NO: 349; or    -   v) the V_(H) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,        98%, 99%, or 100% identical to the amino acid sequence of SEQ ID        NO: 356 and the V_(L) is at least 90%, 91%, 92%, 93%, 94%, 95%,        96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence        of SEQ ID NO: 357; or    -   vi) the V_(H) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, 99%, or 100% identical to the amino acid sequence of        SEQ ID NO: 364 and the V_(L) is at least 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino        acid sequence of SEQ ID NO: 365; or    -   vii) the V_(H) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, 99%, or 100% identical to the amino acid sequence of        SEQ ID NO: 372 and the V_(L) is at least 90%, 91%, 92%, 93%,        94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino        acid sequence of SEQ ID NO: 373.

In general, anti-ICOS antibodies may be administered in an amount in therange of about 10 μg/kg body weight to about 100 mg/kg body weight perdose. In some embodiments, anti-ICOS antibodies may be administered inan amount in the range of about 50 μg/kg body weight to about 5 mg/kgbody weight per dose. In some embodiments, anti-ICOS antibodies may beadministered in an amount in the range of about 100 μg/kg body weight toabout 10 mg/kg body weight per dose. In some embodiments, anti-ICOSantibodies may be administered in an amount in the range of about 100μg/kg body weight to about 20 mg/kg body weight per dose. In someembodiments, anti-ICOS antibodies may be administered in an amount inthe range of about 0.5 mg/kg body weight to about 20 mg/kg body weightper dose.

In some embodiments, the anti-PD-1 antibody provided herein isadministered with an agonist anti-OX40 antibody (such as Medi6469,MedImmune; MOXR0916/RG7888, Roche). In some embodiments, the anti-PD-1antibody provided herein is administered with an anti-CTLA4 antibody(such as ipilimumab, YERVOY®, BMS).

In some embodiments, an additional therapeutic agent is achemotherapeutic agent. Exemplary chemotherapeutic agents that may becombined with the anti-PD-1 antibodies provided herein include, but arenot limited to, capectiabine, cyclophosphamide, dacarbazine,temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin,cisplatin, carboplatin, epirubicin, eribulin, 5-FU, gemcitabine,irinotecan, ixabepilone, methotrexate, mitoxantrone, oxaliplatin,paclitaxel, nab-paclitaxel, ABRAXANE® (protein-bound paclitaxel),pemetrexed, vinorelbine, and vincristine. In some embodiments, ananti-PD-1 antibody provided herein is administered with at least onekinase inhibitor. Nonlimiting exemplary kinase inhibitors includeerlotinib, afatinib, gefitinib, crizotinib, dabrafenib, trametinib,vemurafenib, and cobimetanib.

In some embodiments, the additional therapeutic agent is an IDOinhibitor. Nonlimiting exemplary IDO inhibitors are described, e.g., inUS 2016/0060237; and US 2015/0352206. Nonlimiting exemplary IDOinhibitors include Indoximod (New Link Genetics), INCB024360 (IncyteCorp), 1-methyl-D-tryptophan (New Link Genetics), and GDC-0919(Genentech).

In some embodiments, an anti-PD-1 antibody provided herein isadministered in combination with an immune-modifying drug (IMiD).Nonlimiting exemplary IMiDs include thalidomide, lenalidomide, andpomalidomide.

In some embodiments, an additional therapeutic agent is a cancervaccine. Cancer vaccines have been investigated as a potential approachfor antigen transfer and activation of dendritic cells. In particular,vaccination in combination with immunologic checkpoints or agonists forco-stimulatory pathways have shown evidence of overcoming tolerance andgenerating increased anti-tumor response. A range of cancer vaccineshave been tested that employ different approaches to promoting an immuneresponse against the tumor (see, e.g., Emens L A, Expert Opin EmergDrugs 13(2): 295-308 (2008)). Approaches have been designed to enhancethe response of B cells, T cells, or professional antigen-presentingcells against tumors. Exemplary types of cancer vaccines include, butare not limited to, peptide-based vaccines that employ targetingdistinct tumor antigens, which may be delivered as peptides/proteins oras genetically-engineered DNA vectors, viruses, bacteria, or the like;and cell biology approaches, for example, for cancer vaccine developmentagainst less well-defined targets, including, but not limited to,vaccines developed from patient-derived dendritic cells, autologoustumor cells or tumor cell lysates, allogeneic tumor cells, and the like.

Thus, in certain embodiments, the anti-PD-1 antibodies provided hereinmay be used in combination with a cancer vaccine. Exemplary cancervaccines include, but are not limited to, dendritic cell vaccines,oncolytic viruses, tumor cell vaccines, etc. In some embodiments, suchvaccines augment the anti-tumor response. Examples of cancer vaccinesthat can be used in combination with anti-PD-1 antibodies providedherein include, but are not limited to, MAGE3 vaccine (e.g., formelanoma and bladder cancer), MUC1 vaccine (e.g., for breast cancer),EGFRv3 (such as Rindopepimut, e.g., for brain cancer, includingglioblastoma multiforme), or ALVAC-CEA (e.g., for CEA+ cancers).

Nonlimiting exemplary cancer vaccines also include Sipuleucel-T, whichis derived from autologous peripheral-blood mononuclear cells (PBMCs)that include antigen-presenting cells (see, e.g., Kantoff P W et al., NEngl J Med 363:411-22 (2010)). In Sipuleucel-T generation, the patient'sPBMCs are activated ex vivo with PA2024, a recombinant fusion protein ofprostatic acid phosphatase (a prostate antigen) andgranulocyte-macrophage colony-stimulating factor (an immune-cellactivator). Another approach to a candidate cancer vaccine is togenerate an immune response against specific peptides mutated in tumortissue, such as melanoma (see, e.g., Carreno B M et al., Science348:6236 (2015)). Such mutated peptides may, in some embodiments, bereferred to as neoantigens. As a nonlimiting example of the use ofneoantigens in tumor vaccines, neoantigens in the tumor predicted tobind the major histocompatibility complex protein HLA-A*02:01 areidentified for individual patients with a cancer, such as melanoma.Dendritic cells from the patient are matured ex vivo, then incubatedwith neoantigens. The activated dendritic cells are then administered tothe patient. In some embodiments, following administration of the cancervaccine, robust T-cell immunity against the neoantigen is detectable.

In some such embodiments, the cancer vaccine is developed using aneoantigen. In some embodiments, the cancer vaccine is a DNA vaccine. Insome embodiments, the cancer vaccine is an engineered virus comprising acancer antigen, such as PROSTVAC (rilimogene galvacirepvec/rilimogeneglafolivec). In some embodiments, the cancer vaccine comprisesengineered tumor cells, such as GVAX, which is a granulocyte-macrophagecolony-stimulating factor (GM-CSF) gene-transfected tumor cell vaccine(see, e.g., Nemunaitis, 2005, Expert Rev Vaccines, 4: 259-74).

In some embodiments, an anti-PD-1 antibody described herein isadministered before, concurrently, and/or after a cancer vaccine. Insome embodiments, cancer vaccines developed using neoantigens are usedin combination with the anti-PD-1 antibodies described herein. In somesuch embodiments, the combination is used to treat a cancer with a highmutational burden, such as melanoma, lung, bladder, or colorectalcancer.

In some embodiments, an anti-PD-1 antibody provided herein isadministered in combination with a chimeric antigen receptor T celltherapy (CAR-T therapy).

Diagnostic Uses

Provided herein are methods of using the anti-PD-1 antibodies,polypeptides and polynucleotides for detection, diagnosis and monitoringof a disease, disorder or condition associated with the anti-PD-1antibody epitope expression (either increased or decreased relative to anormal sample, and/or inappropriate expression, such as presence ofexpression in tissues(s) and/or cell(s) that normally lack the epitopeexpression). Provided herein are methods of determining whether apatient will respond to anti-PD-1 antibody therapy.

In some embodiments, the method comprises detecting whether the patienthas cells that express PD-1 using an anti-PD-1 antibody. In someembodiments, the method of detection comprises contacting the samplewith an antibody, polypeptide, or polynucleotide and determining whetherthe level of binding differs from that of a reference or comparisonsample (such as a control). In some embodiments, the method may beuseful to determine whether the antibodies or polypeptides describedherein are an appropriate treatment for the subject.

In some embodiments, the cells or cell/tissue lysate are contacted withan anti-PD-1 antibody and the binding between the antibody and the cellis determined. When the test cells show binding activity as compared toa reference cell of the same tissue type, it may indicate that thesubject would benefit from treatment with an anti-PD-1 antibody. In someembodiments, the test cells are from human tissues. In some embodiments,the test cells are from human blood.

Various methods known in the art for detecting specific antibody-antigenbinding can be used. Exemplary immunoassays which can be conductedinclude fluorescence polarization immunoassay (FPIA), fluorescenceimmunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibitionimmunoassay (NIA), enzyme linked immunosorbent assay (ELISA), andradioimmunoassay (RIA). An indicator moiety, or label group, can beattached to the subject antibodies and is selected so as to meet theneeds of various uses of the method which are often dictated by theavailability of assay equipment and compatible immunoassay procedures.Appropriate labels include, without limitation, radionuclides (forexample ¹²⁵I, ¹³¹I, ³⁵S, ³H, or ³²P), enzymes (for example, alkalinephosphatase, horseradish peroxidase, luciferase, or β-glactosidase),fluorescent moieties or proteins (for example, fluorescein, rhodamine,phycoerythrin, GFP, or BFP), or luminescent moieties (for example, Qdot™nanoparticles supplied by the Quantum Dot Corporation, Palo Alto,Calif.). General techniques to be used in performing the variousimmunoassays noted above are known to those of ordinary skill in theart.

For purposes of diagnosis, the polypeptide including antibodies can belabeled with a detectable moiety including but not limited toradioisotopes, fluorescent labels, and various enzyme-substrate labelsknow in the art. Methods of conjugating labels to an antibody are knownin the art.

In some embodiments, the anti-PD-1 antibodies need not be labeled, andthe presence thereof can be detected using a second labeled antibodywhich binds to the first anti-PD-1 antibody.

In some embodiments, the anti-PD-1 antibody can be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

The anti-PD-1 antibodies and polypeptides can also be used for in vivodiagnostic assays, such as in vivo imaging. Generally, the antibody orthe polypeptide is labeled with a radionuclide (such as ¹¹¹In, ⁹⁹Tc,¹⁴C, ¹³¹I, ¹²⁵I, ³H, or any other radionuclide label, including thoseoutlined herein) so that the cells or tissue of interest can belocalized using immunoscintigraphy.

The antibody may also be used as staining reagent in pathology usingtechniques well known in the art.

In some embodiments, a first antibody is used for a diagnostic and asecond antibody is used as a therapeutic. In some embodiments, the firstand second antibodies are different. In some embodiments, the firstantibody is from a non-human, while the therapeutic is from a human. Insome embodiments, the first and second antibodies can both bind to theantigen at the same time, by binding to separate epitopes.

Kits/Articles of Manufacture

Provided herein are also kits, medicines, compositions, and unit dosageforms for use in any of the methods described herein.

Kits can include one or more containers comprising an anti-PD-1 antibody(or unit dosage forms and/or articles of manufacture). In someembodiments, a unit dosage is provided wherein the unit dosage containsa predetermined amount of a composition comprising an anti-PD-1antibody, with or without one or more additional agents. In someembodiments, such a unit dosage is supplied in single-use prefilledsyringe for injection. In some embodiments, the composition contained inthe unit dosage can comprise saline, sucrose, or the like; a buffer,such as phosphate, or the like; and/or be formulated within a stable andeffective pH range. In some embodiments, the composition can be providedas a lyophilized powder that may be reconstituted upon addition of anappropriate liquid, for example, sterile water. In some embodiments, thecomposition comprises one or more substances that inhibit proteinaggregation, including, but not limited to, sucrose and arginine. Insome embodiments, a composition comprises heparin and/or a proteoglycan.

In some embodiments, the amount of the anti-PD-1 antibody used in theunit dose can be any of the amounts provided herein for the variousmethods and/or compositions described.

In some embodiments, kits further comprise instructions for use in thetreatment of cancer in accordance with any of the methods describedherein. The kit may further comprise a description of selection anindividual suitable or treatment. Instructions supplied in the kits aretypically written instructions on a label or package insert (forexample, a paper sheet included in the kit), but machine-readableinstructions (for example, instructions carried on a magnetic or opticalstorage disk) are also acceptable. In some embodiments, the kit furthercomprises another therapeutic agent. In some embodiments, the additionaltherapeutic agent is an anti-ICOS antibody, e.g., as described herein,or an anti-CTLA4 antibody.

The kits are in suitable packaging. Suitable packaging includes, but isnot limited to, vials, bottles, jars, flexible packaging (for example,sealed Mylar or plastic bags), and the like. Kits may optionally provideadditional components such as buffers and interpretative information.The present application thus also provides articles of manufacture,which include vials (such as sealed vials), bottles, jars, flexiblepackaging, and the like.

EXAMPLES

The examples discussed below are intended to be purely exemplary of theinvention and should not be considered to limit the invention in anyway. The examples are not intended to represent that the experimentsbelow are all or the only experiments performed. Efforts have been madeto ensure accuracy with respect to numbers used (for example, amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

Example 1: Generation of Anti-PD-1 Antibodies

Eight naive human synthetic yeast libraries each of ˜10⁹ diversity weredesigned, generated, and propagated as described previously (see, e.g.,WO2009036379; WO2010105256; WO2012009568; Xu et al., Protein Eng DesSel. 2013 Oct.; 26(10):663-70). For the first 2 rounds of selection, amagnetic bead sorting technique utilizing the Miltenyi MACs system wasperformed, essentially as described (Siegel et al., J Immunol Methods.2004 March; 286(1-2):141-53). Briefly, yeast cells (˜10¹⁰ cells/library)were incubated with 3 ml of 10 nM biotinylated human PD-1-Fc fusionantigen for 15 min at room temperature in FACS wash buffer PBS with 0.1%BSA (biotinylations were done using the EZ-Link Sulfo-NHS-BiotinylationKit, Thermo Scientific, Cat. #21425). After washing once with 50 mlice-cold wash buffer, the cell pellet was resuspended in 40 mL washbuffer, and 500 μl Streptavidin MicroBeads (Miltenyi Biotec, BergischGladbach, Germany, Cat. #130-048-101) were added to the yeast andincubated for 15 min at 4° C. Next, the yeast were pelleted, resuspendedin 5 mL wash buffer, and loaded onto a MACS LS column (Miltenyi Biotec,Bergisch Gladbach, Germany, Cat. #130-042-401). After the 5 mL wasloaded, the column was washed 3 times with 3 ml FACS wash buffer. Thecolumn was then removed from the magnetic field, and the yeast wereeluted with 5 mL of growth media and then grown overnight. The followingthree rounds of sorting were performed using flow cytometry.Approximately 1×10⁸ yeast were pelleted, washed three times with washbuffer, and incubated with 200, 100 or 10 nM biotinylated human PD-1 or10 nM or 1 nM biotinylated human PD-1-Fc fusion antigen from 10 minutesup to 2 hours at room temperature. Incubation time varies with antigenconcentration. Yeast were then washed twice and stained with goatanti-human F(ab′)₂ kappa-FITC diluted 1:100 (Southern Biotech,Birmingham, Ala., Cat. #2062-02) and either streptavidin-Alexa Fluor 633(Life Technologies, Grand Island, N.Y., Cat. #S21375) diluted 1:500, orExtravidin-phycoerthyrin (Sigma-Aldrich, St Louis, Cat. #E4011) diluted1:50, secondary reagents for 15 min at 4° C. After washing twice withice-cold wash buffer, the cell pellets were resuspended in 0.4 mL washbuffer and transferred to strainer-capped sort tubes. Sorting wasperformed using a FACS ARIA sorter (BD Biosciences) and sort gates weredetermined to select only PD-1 binding clones for two rounds. Dependingon the populations, the third round was a negative sort to decreasereagent binders or further positive enrichment. After the final round ofsorting, yeast were plated and individual colonies were picked forcharacterization.

The round 5 or round 6 binding population was used for a light chaindiversification. Heavy chain plasmids were extracted and transformedinto a light chain library with a diversity of 1×10⁶. Selections wereperformed as described above with one round of MACS sorting and threerounds of FACS sorting using various concentrations of biotinylatedantigen.

Affinity Maturation

Binding optimization of naïve and light chain batch shuffle clones wascarried out utilizing diversification of CDRH1 and CDRH2.

CDRH1 and CDRH2 selection: The CDRH3s from clones selected from thelight chain diversification procedure (light chain batch shuffle clones)were recombined into a premade library with CDRH1 and CDRH2 variants ofa diversity of 1×10⁸ and selections were performed using PD-1 antigen,as described above. Affinity pressures were applied by titrating thebiotinylated antigen to sub-nanomolar concentrations and incubating withantibody-presenting yeast.

Antibody Production and Purification

Yeast clones were grown to saturation and then induced for 48 h at 30°C. with shaking. After induction, yeast cells were pelleted and thesupernatants were harvested for purification. IgGs were purified using aProtein A column and eluted with acetic acid, pH 2.0. Fab fragments weregenerated by papain digestion and purified over KappaSelect (GEHealthcare LifeSciences, Cat. #17-5458-01).

Affinity Measurements of PD-1 Antibodies

The affinity of the PD-1 antibodies was determined by measuring theirK_(D) by ForteBio or MSD-SET. ForteBio affinity measurements wereperformed generally as previously described (Estep et al., MAbs. 2013Mar.-Apr.; 5(2):270-8). Briefly, ForteBio affinity measurements wereperformed by loading IgGs on-line onto AHQ sensors. Sensors wereequilibrated off-line in assay buffer for 30 min and then monitoredon-line for 60 seconds for baseline establishment. Sensors with loadedIgGs were exposed to 100 nM antigen for 5 minutes, afterwards they weretransferred to assay buffer for 5 min for off-rate measurement. Kineticswere analyzed using the 1:1 binding model.

Equilibrium affinity measurements performed as previously described(Estep et al., 2013). Solution equilibrium titrations (SET) wereperformed in PBS+0.1% IgG-Free BSA (PBSF) with antigen held constant at50 μM and incubated with 3-to 5-fold serial dilutions of antibodystarting at 10 nM. Antibodies (20 nM in PBS) were coated onto standardbind MSD-ECL plates overnight at 4° C. or at room temperature for 30min. Plates were then blocked for 30 min with shaking at 700 rpm,followed by three washes with wash buffer (PBSF+0.05% Tween 20). SETsamples were applied and incubated on the plates for 150 seconds withshaking at 700 rpm followed by one wash. Antigen captured on a plate wasdetected with 250 ng/mL sulfotag-labeled streptavidin in PBSF byincubation on the plate for 3 min. The plates were washed three timeswith wash buffer and then read on the MSD Sector Imager 2400 instrumentusing 1× Read Buffer T with surfactant. The percent free antigen wasplotted as a function of titrated antibody in Prism and fit to aquadratic equation to extract the K_(D). To improve throughput, liquidhandling robots were used throughout MSD-SET experiments, including SETsample preparation. Table 1 shows the results for the ForteBio affinitymeasurements.

TABLE 1 Human PD-1 Monovalent Mouse PD-1 Monovalent Antibody Affinity(K_(D)) Affinity (K_(D)) mAb 1 (12228)  49 nM 6.3 nM mAb 2 (13405)  73nM 7.2 nM mAb 3 (13406) 3.2 nM 3.5 nM mAb 4 (13407) 3.9 nM 3.2 nM mAb 5(13408) 1.2 nM 5.3 nM

Example 2: Mouse Activated Splenocytes Response to Anti-PD-1 Antibodies

When mouse T cells derived from splenocytes are activated with anti-CD3and anti-CD28 antibodies, they express the cell-surface molecule andsecrete the soluble molecule interferon-gamma (IFNγ). Blockingantibodies to PD-1 cause the production of IFNγ to be increased. Mousesplenocytes were obtained and incubated with soluble anti-CD3 andanti-CD28 antibodies. After 5 days, they were mixed with equal numbersof T cells purified from freshly prepared splenocytes and anti-PD-1antibodies in different concentrations. Positive and negative controlantibodies RMP1-14, 2A3, and C1.18.4 were purchased from BioXCell. After24 hours, supernatants were collected and IFNγ concentrations measuredby cytokine bead array. Anti-PD-1 antibodies disclosed herein were ableto increase the production of IFNγ in a similar way to referenceantibodies. See FIG. 1 . At least antibodies 12191, 13396, 13398, 13399,12195, 13406, 13407, 13408, and 13409 significantly increased IFNγexpression. Antibodies 13396, 13399, 12195, 13406, 13407, and 13408caused the greatest increase in IFNγ expression.

Example 3: Human Activated Peripheral Mononuclear Blood Cells Respond toAnti-PD-1 Antibodies

Whole blood samples activated with Staphylococcal enterotoxin B (SEB)have been shown to respond to an immune checkpoint blockade using ananti-PD-1 antibody, measured by increases of IL-2 secretion (see, e.g.,EP2170959B1). Anti-PD-1 antibodies were prepared in a range ofconcentrations and added to wells of 96 well plates. Whole blood fromfour donors was diluted 1:10 in complete RPMI, and added to the samewells. Plates were incubated at room temperature for 30 minutes beforeaddition of 50 μl SEB per well at a concentration of 40 μg/ml for afinal concentration of 10 μg/ml. Plates were incubated at 37° C. for 4days and supernatants were collected for measurement of secreted IL-2 bycytokine bead array (CBA) assay. Anti-PD-1 antibodies disclosed hereinwere able to increase the production of IL-2 from these cultures to thesame level as reference anti-PD-1 antibodies. FIG. 2 shows the EC₅₀ ofthis increase for each antibody tested, in four healthy human donorsamples.

Example 4: Antibody Blocking of Binding of Mouse PD-L1 and Mouse PD-L2to Mouse PD-1

PD-L1 and PD-L2 are two natural ligands for PD-1 and the interaction ofeither ligand with PD-1 can downregulate an activating response in Tcells. Approved therapeutic antibodies such as nivolumab andpembrolizumab have been shown to block this interaction and downregulatehuman T cell responses, although neither antibody binds to mouse PD-1nor blocks the interaction of mouse PD-1 with mouse PD-L1 or mousePD-L2. Octet analysis on a Forte-Bio instrument was used to assess theeffect of anti-PD-1 antibodies disclosed herein on the interactionbetween mouse PD-L1 and mouse PD-1 and between mouse PD-L2 and mousePD-1. Antibodies were diluted to 10 μg/mL, including an isotype controlantibody for use in background subtraction. Mouse PD-1-Fc fusion wasdiluted to the desired concentrations (e.g., 100 nM). Soluble mouseisotype antibody, to be used as a blocking reagent, was diluted to 250μg/mL. Mouse-PD-1 was coated on to sensors, followed by human Fc.Anti-PD-1 antibodies were added and finally the binding of either mousePD-L1 or mouse PD-L2 was measured. FIG. 3 shows that the anti-PD-1antibodies tested were capable of blocking the binding of both mouseligands to mouse PD-1. In contrast, neither pembrolizumab or nivolumabbound to mouse PD-1 or blocked mouse ligand binding to mouse PD-1.

Example 5: Antibody Blocking of Binding of Human PD-L1 and Human PD-L2to Human PD-1

PD-L1 and PD-L2 are two natural ligands for PD-1 and the interaction ofeither ligand with PD-1 can downregulate an activating response in Tcells. Approved therapeutic antibodies such as nivolumab andpembrolizumab have been shown to block this interaction and downregulatehuman T cell responses. Octet analysis, on a Forte-Bio instrument wasused to assess the effect of anti-PD-1 antibodies disclosed herein onthe interaction between human PD-L1 and human PD-1 and human PD-L2 andhuman PD-1. Antibodies were diluted to 10 μg/mL, including an isotypecontrol antibody for use in background subtraction. Human PD-1-Fc fusionwas diluted to the desired concentrations (e.g., 100 nM). Soluble humanFc, to be used as a blocking reagent, was diluted to 250 μg/mL.Human-PD-1 was coated on to sensors, followed by human Fc. Anti-PD-1antibodies were added and finally the binding of either human PD-L1 orhuman PD-L2 was measured. FIG. 4 shows that the anti-PD-1 antibodiestested were capable of blocking the binding of both human ligands tohuman PD-1, similar to pembrolizumab and nivolumab.

Example 6: In Vivo Tumor Challenge

The ability of anti-PD-1 antibodies to reduce tumor growth in mousemodels was examined by injecting MC38 cells subcutaneously into theflanks of C57BL6/J mice. Mice were randomized into groups with anapproximate 100 mm³ average tumor size on day 11 and injectedintraperitoneally with 200 μg of anti-PD-1 antibody 13407. Repeat doseswere administered on days 14, 18 and 21. Control antibodies, mouse IgG1(MOPC-21), rat IgG2a (2A3), and positive control rat anti-mouse PD-1(RMP1-14) were purchased from BioXcell. Tumor sizes were measured twiceweekly and the experiment was terminated after 24 days. Tumor weights atthe end of the experiment are shown in FIG. 5 and indicate thatanti-PD-1 antibody 13407 reduced tumor growth in mice to a similarextent as the reference anti-PD-1 antibody.

Example 7: Antibody Epitope Mapping

Genes encoding various PD-1 variants, PD-L1, PD-L2 and antibody Fabfragments were synthesized and codon optimized by Atum inc. The humankappa light signal sequence (MGTPAQLLFLLLLWLPDTTG; SEQ ID NO: 400) wasincluded at the 5′ terminus of all genes. Genes were cloned into amammalian expression vector and sequence verified prior to proteinexpression.

Recombinant proteins were expressed in Expi293™ Expression System(Thermo Fisher Scientific) utilizing the manufacturer's kit for media,transfection reagent, feeds and cells. The cells were transfected at 1μg/mL of plasmid DNA to media ratio using Expifectamine™ following themanufacturer's protocol. The cells were cultured in shake flasks in anincubator kept at 37° C. and 5% C02. After 5 days the cultures wereharvested by centrifugation at 3000 rcf for 15 minutes followed byfiltration using a 0.2 μm filter.

Harvested supernatants were subjected to affinity purification. Theproteins either contained a hexa-histidine tag or a Fc tag, whichenabled either Ni-affinity purification or Protein A affinitypurification.

Ni-affinity purifications were carried out on HisTrap™ Excel IMACcolumns (GE Healthcare Life Sciences) using 43 mM sodium phosphate, 0.5M sodium chloride, pH 7.4 as binding buffer. Bound protein was washedwith binding buffer followed by binding buffer containing 20 mMimidazole. Step elutions were performed with binding buffer containing90 mM, 150 mM, and 250 mM imidazole. Protein A affinity purificationswere carried out on HiTrap MabSelect Sure columns (GE Healthcare LifeSciences) using PBS, pH 7.4 as binding buffer. Step elutions wereperformed to elute bound protein using 0.1 M citrate buffer at pH 4.0,pH 3.5 and pH 2.0. All acidic elution fractions were neutralized with 1MTris, pH 9.0 using 20% v/v ratio. All elution fractions were analyzed bySEC-HPLC to determine fractions above target purity (>90% monomeric).For purifications that did not yield fractions above target purity,preparative size exclusion chromatography was performed using aSuperdex200 chromatography column to achieve target purity. Prior tofreezing, purified proteins were buffer exchanged into final buffer(either 20 mM histidine, 150 mM NaCl, pH6.0, or 20 mM HEPES, 150 mMNaCl, pH7.5), keeping them at a pH at least 1 unit away from thepredicted pI of the target protein.

All protein interactions were assessed on a ForteBio OctetRED96instrument. Proteins were diluted in kinetics buffer formulation(PBS+0.1% BSA, 0.02% Tween20, 0.05% sodium azide) with a sample volumewas 220 μl/well. Ligand concentrations were normalized at 10 g/ml andanalyte concentrations at 100 nM. The blocking reagent was employed at aconcentration of 250 μg/ml. Sensor equilibration time was set for 60sec, and sample loading time established at 300 sec. Human Fc blockingtime was set for 300 sec. Analysis of association time was set for 300sec and dissociation time for 600 sec.

For this method, antibodies of interest are oriented and captured onbiosensors derivatized with anti-human (or anti-mouse) Fc antibodies.Binding of target proteins of interest, in solution, to theantibody-loaded sensors is then assayed.

For interactions in which the proteins contain similar species Fcdomains, an additional blocking step is included to minimize directbinding of the target protein to the biosensor independent of thecaptured antibody.

Binding events were confirmed with a Y (yes) if the affinity for a PD-1variant or PD-1 of another species were within approximately 5-foldcompared to affinity for wild-type human PD-1. Affinities that werereduced by >10-fold and <100-fold are indicated as Reduced. Affinitiesreduced by >100-fold are indicated as N (no). Interactions that were notassessed are listed as ND (not determined).

Variants of human PD-1 were produced as extracellular domain (ECD)-Fcfusions with C-terminal His6 tags and used for binding analysis of PD-1ligands as well as Fab fragments of nivolumab, pembrolizumab, and 13407.The results are shown in Table 2.

TABLE 2 Summary of PD-1 binding data PD-1 Variant PD-L1 PD-L2 PD-1(ECD-Fc Fc Fc Nivo Pembro 13407 Species fusions) Fusion Fusion Fab FabFab Human WT Y Y Y Y Y D85G ND ND Y N Y R86L ND ND Y Y Y D85G/R86L ND NDY N Y N33-Q167 Y Y N Y Y K78A N N Y Y Y I126A N Y Y Y Reduced L128AReduced Y Y Y Reduced A132L Y Y Y Y N I134A N Y Y Y N E136A N Y Y YReduced Mouse WT Y Y N N Y H129P ND ND N N N Rat WT ND ND N N N P129H NDND N N Y Dog WT ND ND N N N Cyno WT Y Y Y Y Y

Amino acid substitutions reported to affect binding of pembrolizumabwere assessed for binding to the panel of molecules (Na et al., 2017,Cell Res., 27: 147-150). Pembrolizumab binding to PD-1 is ablated withthe D85G substitution, whereas the binding of nivolumab and 13407 werenot affected. The binding of nivolumab to PD-1 is also diminished whenassessed against the N-terminal truncated variant N33-Q167. Theseresults indicate that 13407 contacts amino acids that are significantlydifferent than those contacted by pembrolizumab and nivolumab.

Additional variants were generated that were previously assessed forPD-L1 and PD-L2 ligands (Lázár-Molnár et al., 2008, PNAS, 105:10483-10488). Binding of nivolumab and pembrolizumab was largelyunaffected by these amino acid substitutions, whereas the bindingaffinity of 13407 was significantly reduced with the I126A, L128A, andE136A substitutions, and eliminated with the A132L and I134Asubstitutions. The data reveal a similar binding profile for 13407 tothat of PD-L1.

The panel of Fab fragments was also assessed for binding to additionalspecies of PD-1. Pembrolizumab and nivolumab fail to bind to mouse, rat,or dog PD-1. In contrast, 13407 binds to mouse PD-1. Surprisingly, 13407fails to bind to rat PD-1 despite the high homology between rat andmouse PD-1. A variant of mouse PD-1 in which residue 129 is replacedwith the corresponding residue from the rat sequence (H129P) wasgenerated and found to ablate binding of 13407. When the reciprocalmutation, P129H, was introduced into the rat sequence, 13407 bound tothe variant. Notable, the dog PD-1 sequence, to which 13407 does notbind, lacks a proline at this position.

In summary, 13407 binds an epitope of PD-1 distinct from the epitopesbound by nivolumab and pembrolizumab.

The disclosure may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the disclosure. Scope of the disclosure is thusindicated by the appended claims rather than by the foregoingdescription, and all changes that come within the meaning and range ofequivalency of the claims are therefore intended to be embraced herein.

TABLE of Sequences SEQ ID NO Description Sequence 1 Human PD-1MQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA amino acidTFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL sequence;PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE UniProtKB/VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS LVLLVWVLAV ICSRAARGTI Swiss-Prot:GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYAT Q15116; 01-IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL AUG-2016 382 Mature humanPGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTS ESFVLNWYRM PD-1 aminoSPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT acid sequenceYLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV (without signalVGVVGGLLGS LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS sequence)VDYGELDFQW REKTPEPPVP CVPEQTEYAT IVFPSGMGTS SPARRGSADGPRSAQPLRPE DGHCSWPL 401 Human PD-1ldspdrpwnp ptfspallvv tegdnatftc sfsntsesfv lnwyrmspsn extracellularqtdklaafpe drsqpgqdcr frvtqlpngr dfhmsvvrar rndsgtylcg domainaislapkaqi keslraelrv terraevpta hpspsprpag qfqggsggDK (ECD)-Fc-His₆THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPEVKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCKVSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGFYPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNVFSCSVMHEAL HNHYTQKSLS LSPGKGSGHH HHHH 385 Human PD-1dspdrpwnpp tfspallvvt egdnatftcs fsntsesfvl nwyrmspsnq D85GECD-tdklaafpeG rsqpgqdcrf rvtqlpngrd fhmsvvrarr ndsgtylcga Fc-His₆islapkaqik eslraelrvt erraevptah pspsprpagq fqtlvggsggDKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPGKGSG HHHHHH 386 Human PD-1dspdrpwnpp tfspallvvt egdnatftcs fsntsesfvl nwyrmspsnq R86L ECD-tdklaafped Lsqpgqdcrf rvtqlpngrd fhmsvvrarr ndsgtylcga Fc-His₆islapkaqik eslraelrvt erraevptah pspsprpagq fqtlvggsggDKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPGKGSG HHHHHH 402 Human PD-1dspdrpwnpp tfspallvvt egdnatftcs fsntsesfvl nwyrmspsnq D85G/R86LtdklaafpeG Lsqpgqdcrf rvtqlpngrd fhmsvvrarr ndsgtylcga ECD-Fc-His₆islapkaqik eslraelrvt erraevptah pspsprpagq fqtlvggsggDKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPGKGSG HHHHHH 387 Human PD-1npptfspall vvtegdnatf tcsfsntses fvlnwyrmsp snqtdklaaf N33-Q167pedrsqpgqd crfrvtqlpn grdfhmsvvr arrndsgtyl cgaislapka ECD-Fc-His₆qikeslrael rvterraevp tahpspsprp agqfqggsgg DKTHTCPPCPAPELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPAPIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGKGSG HHHHHH 388 Human PD-1dspdrpwnpp tfspallvvt egdnatftcs fsntsesfvl nwyrmspsnq K78A ECD-tdAlaafped rsqpgqdcrf rvtqlpngrd fhmsvvrarr ndsgtylcga Fc-His₆islapkaqik eslraelrvt erraevptah pspsprpagq fqtlvggsggDKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPGKGSG HHHHHH 389 Human PD-1dspdrpwnpp tfspallvvt egdnatftcs fsntsesfvl nwyrmspsnq H26A ECD-tdklaafped rsqpgqdcrf rvtqlpngrd fhmsvvrarr ndsgtylcga Fc-His₆Aslapkaqik eslraelrvt erraevptah pspsprpagq fqtlvggsggDKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPGKGSG HHHHHH 390 Human PD-1dspdrpwnpp tfspallvvt egdnatftcs fsntsesfvl nwyrmspsnq L128A ECD-tdklaafped rsqpgqdcrf rvtqlpngrd fhmsvvrarr ndsgtylcga Fc-His₆isAapkaqik eslraelrvt erraevptah pspsprpagq fqtlvggsggDKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPGKGSG HHHHHH 391 Human PD-1dspdrpwnpp tfspallvvt egdnatftcs fsntsesfvl nwyrmspsnq A132L ECD-tdklaafped rsqpgqdcrf rvtqlpngrd fhmsvvrarr ndsgtylcga Fc-His₆islapkLqik eslraelrvt erraevptah pspsprpagq fqtlvggsggDKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPGKGSG HHHHHH 392 Human PD-1dspdrpwnpp tfspallvvt egdnatftcs fsntsesfvl nwyrmspsnq H34A ECD-tdklaafped rsqpgqdcrf rvtqlpngrd fhmsvvrarr ndsgtylcga Fc-His₆islapkaqAk eslraelrvt erraevptah pspsprpagq fqtlvggsggDKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPGKGSG HHHHHH 393 Human PD-1dttgdspdrp wnpptfspal lvvtegdnat ftcsfsntse sfvlnwyrms E136A ECD-psnqtdklaa fpedrsqpgq dcrfrvtqlp ngrdfhmsvv rarrndsgty Fc-His₆lcgaislapk aqikAslrae lrvterraev ptahpspspr pagqfqtlvggsggDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDVSHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNGKEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLTCLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSRWQQGNVFSCS VMHEALHNHY TQKSLSLSPG KGSGHHHHHH 2 Mouse PD-1;MWVRQVPWSF TWAVLQLSWQ SGWLLEVPNG PWRSLTFYPA WLTVSEGANA UniProtKB/TFTCSLSNWS EDLMLNWNRL SPSNQTEKQA AFCNGLSQPV QDARFQIIQL Swiss-Prot:PNRHDFHMNI LDTRRNDSGI YLCGAISLHP KAKIEESPGA ELVVTERILE Q02242; 01-TSTRYPSPSP KPEGRFQGMV IGIMSALVGI PVLLLLAWAL AVFCSTSMSE AUG-2016ARGAGSKDDT LKEEPSAAPV PSVAYEELDF QGREKTPELP TACVHTEYATIVFTEGLGAS AMGRRGSADG LQGPRPPRHE DGHCSWPL 383 Mature mouseSGWLLEVPNG PWRSLTFYPA WLTVSEGANA TFTCSLSNWS EDLMLNWNRL PD-1 aminoSPSNQTEKQA AFCNGLSQPV QDARFQIIQL PNRHDFHMNI LDTRRNDSGI acid sequenceYLCGAISLHP KAKIEESPGA ELVVTERILE TSTRYPSPSP KPEGRFQGMV (without signalIGIMSALVGI PVLLLLAWAL AVFCSTSMSE ARGAGSKDDT LKEEPSAAPV sequence)PSVAYEELDF QGREKTPELP TACVHTEYAT IVFTEGLGAS AMGRRGSADGLQGPRPPRHE DGHCSWPL 403 Mouse PD-1levpngpwrs ltfypawltv seganatftc slsnwsedlm lnwnrlspsn ECD-Fc-His₆qtekqaafcn glsqpvqdar fqiiqlpnrh dfhmnildtr rndsgiylcgaislhpkaki eespgaelvv teriletstr ypspspkpeg rfqggsggDKTHTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPEVKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCKVSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGFYPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNVFSCSVMHEAL HNHYTQKSLS LSPGKGSGHH HHHH 394 Mouse H129Plevpngpwrs ltfypawltv seganatftc slsnwsedlm lnwnrlspsn PD-1 ECD-Fc-qtekqaafcn glsqpvqdar fqiiqlpnrh dfhmnildtr rndsgiylcg His₆aislPpkaki eespgaelvv teriletstr ypspspkpeg rfqggsggDKTHTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPEVKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCKVSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGFYPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNVFSCSVMHEAL HNHYTQKSLS LSPGKGSGHH HHHH 3 CynomolgusMQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA Monkey PD-1;TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL UniProtKB/PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE Swiss-Prot:VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS LVLLVWVLAV ICSRAARGTI B0LAJ3; 01-GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYAT AUG-2016IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL 384 MaturePGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTS ESFVLNWYRM cynomolgusSPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT monkey PD-1YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV (without signalVGVVGGLLGS LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS sequence)VDYGELDFQW REKTPEPPVP CVPEQTEYAT IVFPSGMGTS SPARRGSADGPRSAQPLRPE DGHCSWPL 404 CynomolgusLESPDR PWNAPTFSPA LLLVTEGDNA TFTCSFSNAS ESFVLNWYRM monkey PD-1SPSNQTDKLA AFPEDRSQPG QDCRFRVTRL PNGRDFHMSV VRARRNDSGT ECD-Fc-His₆YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQGGSGGDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSHEDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKEYKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM TKNQVSLTCLVKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQQGNVFSCSVM HEALHNHYTQ KSLSLSPGKG SGHHHHHH 395 Rat PD-1;MWVQQVPWSF TWAVLQLSWQ SGWLLEVLNK PWRPLTFSPT WLTVSEGANA NCBI Ref.TFTCSFSNWS EDLKLNWYRL SPSNQTEKQA AFCNGYSQPV RDARFQIVQL NP_001100397.1;PNGHDFHMNI LDARRNDSGI YLCGAISLPP KAQIKESPGA ELVVTERILE 02-OCT-TPTRYPRPSP KPEGQFQGLV IVIMSVLVGI PVLLLLAWAL AAFCSTGMSE 2017AREAGRKEDP PKEAHAAAPV PSVAYEELDF QGREKTPEPA PCVHTEYATIVFTEGLDASA IGRRGSADGP QGPRPPRHED GHCSWPL 396 Mature rat PD-LEVLNKPWRP LTFSPTWLTV SEGANATFTC SFSNWSEDLK LNWYRLSPSN 1 amino acidQTEKQAAFCN GYSQPVRDAR FQIVQLPNGH DFHMNILDAR RNDSGIYLCG sequenceAISLPPKAQI KESPGAELVV TERILETPTR YPRPSPKPEG QFQGLVIVIM (without signalSVLVGIPVLL LLAWALAAFC STGMSEAREA GRKEDPPKEA HAAAPVPSVA sequence)YEELDFQGRE KTPEPAPCVH TEYATIVFTE GLDASAIGRR GSADGPQGPR PPRHEDGHCS WPL405 Rat PD-1 levlnkpwrp ltfsptwltv seganatftc sfsnwsedlk lnwyrlspsnECD-Fc-His₆ qtekqaafcn gysqpvrdar fqivqlpngh dfhmnildar rndsgiylcgaislppkaqi kespgaelvv teriletptr yprpspkpeg qfqggsggDKTHTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPEVKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCKVSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGFYPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNVFSCSVMHEAL HNHYTQKSLS LSPGKGSGHH HHHH 397 Rat P129Hlevlnkpwrp ltfsptwltv seganatftc sfsnwsedlk lnwyrlspsn PD-1 ECD-Fc-qtekqaafcn gysqpvrdar fqivqlpngh dfhmnildar rndsgiylcg His₆aislHpkaqi kespgaelvv teriletptr yprpspkpeg qfqggsggDKTHTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPEVKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCKVSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGFYPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNVFSCSVMHEAL HNHYTQKSLS LSPGKGSGHH HHHH 398 Dog PD-1MGSRRGPWPL VWAVLQLGWW PGWLLDSPDR PWSPLTFSPA QLTVQEGENA amino acidTFTCSLADIP DSFVLNWYRL SPRNQTDKLA AFQEDRIEPG RDRRFRVTRL sequence;PNGRDFHMSI VAARLNDSGI YLCGAIYLPP NTQINESPRA ELSVTERTLE NCBI Ref.PPTQSPSPPP RLSGQLQGLV IGVTSVLVGV LLLLLLTWVL AAVFPRATRG NP_001301026.1ACVCGSEDEP LKEGPDAAPV FTLDYGELDF QWREKTPEPP APCAPEQTEYATIVFPGRPA SPGRRASASS LQGAQPPSPE DGPGLWPP 399 Mature dogLDSPDRPWSP LTFSPAQLTV QEGENATFTC SLADIPDSFV LNWYRLSPRN PD-1 aminoQTDKLAAFQE DRIEPGRDRR FRVTRLPNGR DFHMSIVAAR LNDSGIYLCG acid sequenceAIYLPPNTQI NESPRAELSV TERTLEPPTQ SPSPPPRLSG QLQGLVIGVT (without signalSVLVGVLLLL LLTWVLAAVF PRATRGACVC GSEDEPLKEG PDAAPVFTLD sequence)YGELDFQWRE KTPEPPAPCA PEQTEYATIV FPGRPASPGR RASASSLQGA QPPSPEDGPG LWPP406 Dog PD-1 LDSPDR PWSPLTFSPA QLTVQEGENATFTCSLADIP DSFVLNWYRLECD-Fc-His₆ SPRNQTDKLA AFQEDRIEPG RDRRFRVTRL PNGRDFHMSI VAARLNDSGIYLCGAIYLPP NTQINESPRA ELSVTERTLE PPTQSPSPPP RLSGQLQGGSGGDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSHEDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKEYKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM TKNQVSLTCLVKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQQGNVFSCSVM HEALHNHYTQ KSLSLSPGKG SGHHHHHH 4 12228QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYYMHWVRQA PGQGLEWMGI VH SequenceINPSGGSTSY AQKFQGRVTM TRDTSTSTVY MELSSLRSED TAVYYCARGGTYYDYTYWGQ GTLVTVSS 5 12228 YTFTSYYMH HCDR1 6 12228 IINPSGGSTSYAQKFQGHCDR2 7 12228 ARGGTYYDYTY HCDR3 8 12228DIQMTQSPST LSASVGDRVT ITCRASQSIS SWLAWYQQKP GKAPKLLIYE VL SequenceASSLESGVPS RFSGSGSGTE FTLTISSLQP DDFATYYCQQ YNSFPPTFGG GTKVEIK 9 12228RASQSISSWLA LCDR1 10 12228 EASSLES LCDR2 11 12228 QQYNSFPPT LCDR3 1213406 QVQLVQSGAEVKKPGASVKVSCKASGYTFDQYYMH VH SequenceWVRQAPGQGLEWMGIINPSGGSTAYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGTYYDYTYWGQGTLVTVSS 13 13406 YTFDQYYMHHCDR1 14 13406 IINPSGGSTAYAQKFQG HCDR2 15 13406 ARGGTYYDYTY HCDR3 1613406 DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGK VL SequenceAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYY CQQYNSFPPTFGGGTKVEIK 1713406 RASQSISSWLA LCDR1 18 13406 EASSLES LCDR2 19 13406 QQYNSFPPT LCDR320 13407 QVQLVQSGAEVKKPGASVKVSCKASGYTFPSYYMHWVRQAPGQGL VH SequenceEWMGIINPEGGSTAYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGTYYDYTYWGQGTLVTVSS 21 13407 YTFPSYYMH HCDR1 22 13407IINPEGGSTAYAQKFQG HCDR2 23 13407 ARGGTYYDYTY HCDR3 24 13407DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKP VL SequenceGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDF ATYYCQQYNSFPPTFGGGTKVEIK 2513407 RASQSISSWLA LCDR1 26 13407 EASSLES LCDR2 27 13407 QQYNSFPPT LCDR328 13408 QVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYMHWVRQ VH SequenceAPGQGLEWMGIINPSGGVTAYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGTYYDYTYWGQGTLVTVSS 29 13408 YTFSDYYMH HCDR1 3013408 IINPSGGVTAYAQKFQG HCDR2 31 13408 ARGGTYYDYTY HCDR3 32 13408DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQ VL SequenceQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISS LQPDDFATYYCQQYNSFPPTFGGGTKVEIK33 13408 RASQSISSWLA LCDR1 34 13408 EASSLES LCDR2 35 13408 QQYNSFPPTLCDR3 36 13409 QVQLVQSGAEVKKPGASVKVSCKASGYTFESYYMHWVRQ VH SequenceAPGQGLEWMGIINPSGGVTAYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGTYYDYTYWGQGTLVTVSS 37 13409 YTFESYYMH HCDR1 3813409 IINPSGGVTAYAQKFQG HCDR2 39 13409 ARGGTYYDYTY HCDR3 40 13409DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQ VL SequenceQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISS LQPDDFATYYCQQYNSFPPTFGGGTKVEIK41 13409 RASQSISSWLA LCDR1 42 13409 EASSLES LCDR2 43 13409 QQYNSFPPTLCDR3 44 13396 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGSequence IINPDAGSTAYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDQGHYYGMGVWGQGTTVTVSS 45 13396 YTFTSYYMH HCDR1 46 13396IINPDAGSTAYAQKFQG HCDR2 47 13396 ARDQGHYYGMGV HCDR3 48 13396 VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIY SequenceEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQHNSYPPTF GGGTKVEIK 4913396 LCDR1 RASQSISSWLA 50 13396 LCDR2 EASSLES 51 13396 LCDR3 QQHNSYPPT52 13398 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFGEYYMHWVRQAPGQGLEWMG SequenceIINPSEGSTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR DQGHYYGMGVWGQGTTVTVSS53 13398 YTFGEYYMH HCDR1 54 13398 IINPSEGSTGYAQKFQG HCDR2 55 13398 HCDR3ARDQGHYYGMGV 56 13398 VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSL SequenceESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQHNSYPPTFGGGTKVEIK 57 13398 LCDR1RASQSISSWLA 58 13398 LCDR2 EASSLES 59 13398 LCDR3 QQHNSYPPT 60 13399 VHQVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYMHWVRQAPGQGLEWMG SequenceIINPSAGSTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR DQGHYYGMGVWGQGTTVTVSS61 13399 YTFSDYYMH HCDR1 62 13399 IINPSAGSTGYAQKFQG HCDR2 63 13399 HCDR3ARDQGHYYGMGV 64 13399 VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIY SequenceEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQHNSYPPTF GGGTKVEIK 6513399 LCDR1 RASQSISSWLA 66 13399 LCDR2 EASSLES 67 13399 LCDR3 QQHNSYPPT68 13401 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFNSYYMSWVRQAPGQGLEWMG SequenceIIDPSKGSTAYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR HEYGMDVWGQGTTVTVSS 6913401 YTFNSYYMS HCDR1 70 13401 IIDPSKGSTAYAQKFQG HCDR2 71 13401 HCDR3ARHEYGMDV 72 13401 VL DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYSequence EASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSFPPTF GGGTKVEIK 7313401 LCDR1 RASQSISSWLA 74 13401 LCDR2 EASSLES 75 13401 LCDR3 QQYNSFPPT76 13402 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTHYYMSWVRQAPGQGLEWMG SequenceMINPEGGSTAYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR HEYGMDVWGQGTTVTVSS 7713402 YTFTHYYMS HCDR1 78 13402 MINPEGGSTAYAQKFQG HCDR2 79 13402 HCDR3ARHEYGMDV 80 13402 VL DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYSequence EASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSFPPTF GGGTKVEIK 8113402 LCDR1 RASQSISSWLA 82 13402 LCDR2 EASSLES 83 13402 LCDR3 QQYNSFPPT84 13403 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFDSYYMSWVRQAPGQGLEWMG SequenceMINPSVGSTAYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR HEYGMDVWGQGTTVTVSS 8513403 YTFDSYYMS HCDR1 86 13403 MINPSVGSTAYAQKFQG HCDR2 87 13403 HCDR3ARHEYGMDV 88 13403 VL DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYSequence EASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSFPPTF GGGTKVEIK 8913403 LCDR1 RASQSISSWLA 90 13403 LCDR2 EASSLES 91 13403 LCDR3 QQYNSFPPT92 13404 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFASYAMSWVRQAPGQGLEWMG SequenceIIFPGGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR HEYGMDVWGQGTTVTVSS 9313404 YTFASYAMS HCDR1 94 13404 IIFPGGGSTSYAQKFQG HCDR2 95 13404 HCDR3ARHEYGMDV 96 13404 VL DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYSequence EASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSFPPTF GGGTKVEIK 9713404 LCDR1 RASQSISSWLA 98 13404 LCDR2 EASSLES 99 13404 LCDR3 QQYNSFPPT100 13405 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFASYYMGWVRQAPGQGLEWMG SequenceIINPAGGSTAYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR HEYGMDVWGQGTTVTVSS 10113405 YTFASYYMG HCDR1 102 13405 IINPAGGSTAYAQKFQG HCDR2 103 13405 HCDR3ARHEYGMDV 104 13405 VL DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYSequence EASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSFPPTF GGGTKVEIK 10513405 LCDR1 RASQSISSWLA 106 13405 LCDR2 EASSLES 107 13405 LCDR3QQYNSFPPT 108 12191 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGSequence IINPGGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDQGHYYGMGVWGQGTTVTVSS 109 12191 YTFTSYYMH HCDR1 110 12191IINPGGGSTSYAQKFQG HCDR2 111 12191 HCDR3 ARDQGHYYGMGV 112 12191 VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIY SequenceEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQHNSYPPTF GGGTKVEIK 11312191 LCDR1 RASQSISSWLA 114 12191 LCDR2 EASSLES 115 12191 LCDR3QQHNSYPPT 116 12195 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMSWVRQAPGQGLEWMGSequence IINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARHEYGMDVWGQGTTVTVSS 117 12195 YTFTSYYMS HCDR1 118 12195 IINPSGGSTSYAQKFQGHCDR2 119 12195 HCDR3 ARHEYGMDV 120 12195 VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIY SequenceEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSFPPTF GGGTKVEIK 12112195 LCDR1 RASQSISSWLA 122 12195 LCDR2 EASSLES 123 12195 LCDR3QQYNSFPPT 124 12535 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYLMSWVRQAPGKGLEWVSSequence AISGSGKSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVYYGMPYWGQGTLVTVSS 125 12535 FTFSHYLMS HCDR1 126 12535 AISGSGKSTYYADSVKGHCDR2 127 12535 HCDR3 AKVYYGMPY 128 12535 VLDIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIY SequenceAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQASDVPWTF GGGTKVEIK 12912535 LCDR1 RASQGIDSWLA 130 12535 LCDR2 AASSLQS 131 12535 LCDR3QQASDVPWT 132 12536 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYLMSWVRQAPGKGLEWVSSequence AIGGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVYYGMPYWGQGTLVTVSS 133 12536 FTFSQYLMS HCDR1 134 12536 AIGGSGASTYYADSVKGHCDR2 135 12536 HCDR3 AKVYYGMPY 136 12536 VLDIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIY SequenceAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQASDVPWTF GGGTKVEIK 13712536 LCDR1 RASQGIDSWLA 138 12536 LCDR2 AASSLQS 139 12536 LCDR3QQASDVPWT 140 12541 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFRSYMMSWVRQAPGKGLEWVSSequence AISGSGRDTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVYYGMPYWGQGTLVTVSS 141 12541 FTFRSYMMS HCDR1 142 12541 AISGSGRDTYYADSVKGHCDR2 143 12541 HCDR3 AKVYYGMPY 144 12541 VLDIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIY SequenceAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQASDVPWTF GGGTKVEIK 14512541 LCDR1 RASQGIDSWLA 146 12541 LCDR2 AASSLQS 147 12541 LCDR3QQASDVPWT 148 12543 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYMMSWVRQAPGKGLEWVSSequence GISGSGGETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVYYGMPYWGQGTLVTVSS 149 12543 FTFSQYMMS HCDR1 150 12543 GISGSGGETYYADSVKGHCDR2 151 12543 HCDR3 AKVYYGMPY 152 12543 VLDIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIY SequenceAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQASDVPWTF GGGTKVEIK 15312543 LCDR1 RASQGIDSWLA 154 12543 LCDR2 AASSLQS 155 12543 LCDR3QQASDVPWT 156 12544 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYLMSWVRQAPGKGLEWVSSequence AISGSGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVYYGMPYWGQGTLVTVSS 157 12544 FTFSSYLMS HCDR1 158 12544 AISGSGSSTYYADSVKGHCDR2 159 12544 HCDR3 AKVYYGMPY 160 12544 VLDIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIY SequenceAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQASDVPWTF GGGTKVEIK 16112544 LCDR1 RASQGIDSWLA 162 12544 LCDR2 AASSLQS 163 12544 LCDR3QQASDVPWT 164 12545 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYLMSWVRQAPGKGLEWVSSequence AISGSGGQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVYYGMPYWGQGTLVTVSS 165 12545 FTFSHYLMS HCDR1 166 12545 AISGSGGQTYYADSVKGHCDR2 167 12545 HCDR3 AKVYYGMPY 168 12545 VLDIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIY SequenceAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQASDVPWTF GGGTKVEIK 16912545 LCDR1 RASQGIDSWLA 170 12545 LCDR2 AASSLQS 171 12545 LCDR3QQASDVPWT 172 12549 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSQYSMSWVRQAPGKGLEWVSAISGG SequenceGGQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVYYGMPYWGQ GTLVTVSS 17312549 FTFSQYSMS HCDR1 174 12549 AISGGGGQTYYADSVKG HCDR2 175 12549 HCDR3AKVYYGMPY 176 12549 VL DIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYSequence AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQASDVPWTF GGGTKVEIK 17712549 LCDR1 RASQGIDSWLA 178 12549 LCDR2 AASSLQS 179 12549 LCDR3QQASDVPWT 180 12550 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSLYAMSWVRQAPGKGLEWVSSequence AISGSGGQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVYYGMPYWGQGTLVTVSS 181 12550 FTFSLYAMS HCDR1 182 12550 AISGSGGQTYYADSVKGHCDR2 183 12550 HCDR3 AKVYYGMPY 184 12550 VLDIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIY SequenceAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQASDVPWTF GGGTKVEIK 18512550 LCDR1 RASQGIDSWLA 186 12550 LCDR2 AASSLQS 187 12550 LCDR3QQASDVPWT 188 12553 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFGEYAISWVRQAPGQGLEWMGSequence LIIPIFGTAQYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGASDGETGRLDLWGRGTLVTVSS 189 12553 GTFGEYAIS HCDR1 190 12553LIIPIFGTAQYAQKFQG HCDR2 191 12553 HCDR3 ARGASDGETGRLDL 192 12553 VLDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQP SequencePKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSY SLPFTFGGGTKVEIK 19312553 LCDR1 KSSQSVLYSSNNKNYLA 194 12553 LCDR2 WASTRES 195 12553 LCDR3QQSYSLPFT 196 12554 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGSequence LIIPAFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGASDGETGRLDLWGRGTLVTVSS 197 12554 GTFSSYAIS HCDR1 198 12554LIIPAFGTANYAQKFQG HCDR2 199 12554 HCDR3 ARGASDGETGRLDL 200 12554 VLDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQP SequencePKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSY SLPFTFGGGTKVEIK 20112554 LCDR1 KSSQSVLYSSNNKNYLA 202 12554 LCDR2 WASTRES 203 12554 LCDR3QQSYSLPFT 204 12562 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGSequence VIIPIFGEANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGASDGETGRLDLWGRGTLVTVSS 205 12562 GTFSSYAIS HCDR1 206 12562VIIPIFGEANYAQKFQG HCDR2 207 12562 HCDR3 ARGASDGETGRLDL 208 12562 VLDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQP SequencePKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSY SLPFTFGGGTKVEIK 20912562 LCDR1 KSSQSVLYSSNNKNYLA 210 12562 LCDR2 WASTRES 211 12562 LCDR3QQSYSLPFT 212 12563 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFGSYAISWVRQAPGQGLEWMGSequence VIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGASDGETGRLDLWGRGTLVTVSS 213 12563 GTFGSYAIS HCDR1 214 12563VIIPIFGTANYAQKFQG HCDR2 215 12563 HCDR3 ARGASDGETGRLDL 216 12563 VLDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQP SequencePKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSY SLPFTFGGGTKVEIK 21712563 LCDR1 KSSQSVLYSSNNKNYLA 218 12563 LCDR2 WASTRES 219 12563 LCDR3QQSYSLPFT 220 12564 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSQYAISWVRQAPGQGLEWMGSequence VIIPSFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGASDGETGRLDLWGRGTLVTVSS 221 12564 GTFSQYAIS HCDR1 222 12564VIIPSFGTANYAQKFQG HCDR2 223 12564 HCDR3 ARGASDGETGRLDL 224 12564 VLDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQP SequencePKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSY SLPFTFGGGTKVEIK 22512564 LCDR1 KSSQSVLYSSNNKNYLA 226 12564 LCDR2 WASTRES 227 12564 LCDR3QQSYSLPFT 228 12565 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFGEYAISWVRQAPGQGLEWMGSequence LIIPIFGTAQYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGASDGETGRLDLWGRGTLVTVSS 229 12565 GTFGEYAIS HCDR1 230 12565LIIPIFGTAQYAQKFQG HCDR2 231 12565 HCDR3 ARGASDGETGRLDL 232 12565 VLDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQP SequencePKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSY SLPFTFGGGTKVEIK 23312565 LCDR1 KSSQSVLYSSNNKNYLA 234 12565 LCDR2 WASTRES 235 12565 LCDR3QQSYSLPFT 236 12571 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFGSSAISWVRQAPGQGLEWMGSequence GIIPLFGTAAYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDGPGYSSSWYLDVWGQGTMVTVSS 237 12571 GTFGSSAIS HCDR1 238 12571GIIPLFGTAAYAQKFQG HCDR2 239 12571 HCDR3 ARDGPGYSSSWYLDV 240 12571 VLEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY SequenceDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQGYALPITF GGGTKVEIK 24112571 LCDR1 RASQSVSSYLA 242 12571 LCDR2 DASNRAT 243 12571 LCDR3QQGYALPIT 244 12572 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSEYAISWVRQAPGQGLEWMGSequence GIIPIFGTAVYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDGPGYSSSWYLDVWGQGTMVTVSS 245 12572 GTFSEYAIS HCDR1 246 12572GIIPIFGTAVYAQKFQG HCDR2 247 12572 HCDR3 ARDGPGYSSSWYLDV 248 12572 VLEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY SequenceDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQGYALPITF GGGTKVEIK 24912572 LCDR1 RASQSVSSYLA 250 12572 LCDR2 DASNRAT 251 12572 LCDR3QQGYALPIT 252 12576 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGSequence GIIPIFGTAVYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDGPGYSSSWYLDVWGQGTMVTVSS 253 12576 GTFSSYAIS HCDR1 254 12576GIIPIFGTAVYAQKFQG HCDR2 255 12576 HCDR3 ARDGPGYSSSWYLDV 256 12576 VLEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY SequenceDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQGYALPITF GGGTKVEIK 25712576 LCDR1 RASQSVSSYLA 258 12576 LCDR2 DASNRAT 259 12576 LCDR3QQGYALPIT 260 12583 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSSVISWVRQAPGQGLEWMGSequence GIIPIFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDGPGYSSSWYLDVWGQGTMVTVSS 261 12583 GTFSSSVIS HCDR1 262 12583GIIPIFGTATYAQKFQG HCDR2 263 12583 HCDR3 ARDGPGYSSSWYLDV 264 12583 VLEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY SequenceDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQGYALPITF GGGTKVEIK 26512583 LCDR1 RASQSVSSYLA 266 12583 LCDR2 DASNRAT 267 12583 LCDR3QQGYALPIT 268 12584 VH QVQLVQSGAEVKKPGASVKVSCKASGGTFDSYVISWVRQAPGQGLEWMGSequence GIIPGFGVANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDGPGYSSSWYLDVWGQGTMVTVSS 269 12584 GTFDSYVIS HCDR1 270 12584GIIPGFGVANYAQKFQG HCDR2 271 12584 HCDR3 ARDGPGYSSSWYLDV 272 12584 VLEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY SequenceDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQGYALPITF GGGTKVEIK 27312584 LCDR1 RASQSVSSYLA 274 12584 LCDR2 DASNRAT 275 12584 LCDR3QQGYALPIT 276 11613 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSequence AISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVYYGMPYWGQGTLVTVSS 277 11613 FTFSSYAMS HCDR1 278 11613 AISGSGGSTYYADSVKGHCDR2 279 11613 HCDR3 AKVYYGMPY 280 11613 VLDIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIY SequenceAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQASDVPWTF GGGTKVEIK 28111613 LCDR1 RASQGIDSWLA 282 11613 LCDR2 AASSLQS 283 11613 LCDR3QQASDVPWT 284 11645 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGSequence GIIPIFGTASYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDGPGYSSSWYLDVWGQGTMVTVSS 285 11645 GTFSSYAIS HCDR1 286 11645GIIPIFGTASYAQKFQG HCDR2 287 11645 HCDR3 ARDGPGYSSSWYLDV 288 11645 VLEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY SequenceDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQGYALPITF GGGTKVEIK 28911645 LCDR1 RASQSVSSYLA 290 11645 LCDR2 DASNRAT 291 11645 LCDR3QQGYALPIT 292 11606 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGSequence IINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARAGGYSYSWGGSNIWGQGTMVTVSS 293 11606 YTFTSYYMH HCDR1 294 11606IINPSGGSTSYAQKFQG HCDR2 295 11606 HCDR3 ARAGGYSYSWGGSNI 296 11606 VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIY SequenceKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQDGSFPYTF GGGTKVEIK 29711606 LCDR1 RASQSISSWLA 298 11606 LCDR2 KASSLES 299 11606 LCDR3QQDGSFPYT 300 12220 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGSequence IINPGGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDPRYTTLTGSYYYGMDVWGQGTTVTVSS 301 12220 YTFTSYYMH HCDR1 302 12220IINPGGGSTSYAQKFQG HCDR2 303 12220 HCDR3 ARDPRYTTLTGSYYYGMDV 304 12220 VLDIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPKLLIY SequenceAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAVNFPPIT FGGGTKVEIK 30512220 LCDR1 RASQDISSWLA 306 12220 LCDR2 AASSLQS 307 12220 LCDR3QQAVNFPPIT 308 11624 VHQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMG SequenceWINPNSGGTKYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR EASWLPGSLDVWGKGTTVTVSS309 11624 YTFTGYYMH HCDR1 310 11624 WINPNSGGTKYAQKFQG HCDR2 31111624 HCDR3 AREASWLPGSLDV 312 11624 VLDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQP SequencePKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYD NFPITFGGGTKVEIK 31311624 LCDR1 KSSQSVLYSSNNKNYLA 314 11624 LCDR2 WASTRES 315 11624 LCDR3QQYDNFPIT 316 12190 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGSequence IINPGGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDPRYTTLTGSYYYGMDVWGQGTTVTVSS 317 12190 YTFTSYYMH HCDR1 318 12190IINPGGGSTSYAQKFQG HCDR2 319 12190 HCDR3 ARDPRYTTLTGSYYYGMDV 320 12190 VLDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY SequenceAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSANTPPWT FGGGTKVEIK 32112190 LCDR1 RASQSISSYLN 322 12190 LCDR2 AASSLQS 323 12190 LCDR3QQSANTPPWT 324 37A10 heavy EVQLVESGGGLVKPGGSLKLSCAASGF chain variableTFSDYWMDWVRQAPGKGLEWVGNIDED region GSITEYSPFVKGRFTISRDNVKNTLYLQMNSVKSEDTATYYCTRWGRFGFDSWG QGTLVTVSS 325 37A10 lightDIVMTQSPSSLAVSAGDRVTINCKSSQ chain variable SLLSGSFNYLTWYQQKTGQAPKLLIFYregion ASTRHTGVPDRFMGSGSGTDFTLTINS FQTEDLGDYYCHHHYNAPPTFGPGTKL ELR 32637A10 VH GFTFSDYWMD CDR1 327 37A10 VH NIDEDGSITEYSPFVKG CDR2 32837A10 VH WGRFGFDS CDR3 329 37A10 VL KSSQSLLSGSFNYLT CDR1 330 37A10 VLYASTRHT CDR2 331 37A10 VL HHHYNAPPT CDR3 332 37A10S713EVQLVESGGG LVQPGGSLRL SCAASGFTFS DYWMDWVRQA PGKGLVWVSN heavy chainIDEDGSITEY SPFVKGRFTI SRDNAKNTLY LQMNSLRAED TAVYYCTRWG variable regionRFGFDSWGQG TLVTVSS 333 37A10S713DIVMTQSPDS LAVSLGERAT INCKSSQSLL SGSFNYLTWY QQKPGQPPKL light chainLIFYASTRHT GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCHHHYNAPP variable regionTFGPGTKVDI K 334 37A10S713 GFTFSDYWMD VH CDR1 335 37A10S713NIDEDGSITEYSPFVKG VHCDR2 336 37A10S713 WGRFGFDS VH CDR3 337 37A10S713KSSQSLLSGSFNYLT VL CDR1 338 37A10S713 YASTRHT VL CDR2 339 37A10S713HHHYNAPPT VL CDR3 340 37A10S714EVQLVESGGG LVQPGGSLRL SCAASGFTFS DYWMDWVRQA PGKGLVWVSN heavy chainIDEDGSITEY SPFVKGRFTI SRDNAKNTLY LQMNSLRAED TAVYYCTRWG variable regionRFGFDSWGQG TLVTVSS 341 37A10S714DIVMTQSPDS LAVSLGERAT INCKSSQSLL SGSFNYLTWY QQKPGQPPKL light chainLIFYASTRET GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCHHHYNAPP variable regionTFGPGTKVDI K 342 37A10S714 GFTFSDYWMD VH CDR1 343 37A10S714NIDEDGSITEYSPFVKG VHCDR2 344 37A10S714 WGRFGFDS VH CDR3 345 37A10S714KSSQSLLSGSFNYLT VL CDR1 346 37A10S714 YASTRET VL CDR2 347 37A10S714HHHYNAPPT VL CDR3 348 37A10S715EVQLVESGGG LVQPGGSLRL SCAASGFTFS DYWMDWVRQA PGKGLVWVSN heavy chainIDEDGSITEY SPFVKGRFTI SRDNAKNTLY LQMNSLRAED TAVYYCTRWG variable regionRFGFDSWGQG TLVTVSS 349 37A10S715DIVMTQSPDS LAVSLGERAT INCKSSQSLL SGSFNYLTWY QQKPGQPPKL light chainLIFYASTRQT GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCHHHYNAPP variable regionTFGPGTKVDI K 350 37A10S715 GFTFSDYWMD VH CDR1 351 37A10S715NIDEDGSITEYSPFVKG VHCDR2 352 37A10S715 WGRFGFDS VH CDR3 353 37A10S715KSSQSLLSGSFNYLT VL CDR1 354 37A10S715 YASTRQT VL CDR2 355 37A10S715HHHYNAPPT VL CDR3 356 37A10S716EVQLVESGGG LVQPGGSLRL SCAASGFTFS DYWMDWVRQA PGKGLVWVSN heavy chainIDESGSITEY SPFVKGRFTI SRDNAKNTLY LQMNSLRAED TAVYYCTRWG variable regionRFGFDSWGQG TLVTVSS 357 37A10S716DIVMTQSPDS LAVSLGERAT INCKSSQSLL SGSFNYLTWY QQKPGQPPKL light chainLIFYASTRHT GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCHHHYNAPP variable regionTFGPGTKVDI K 358 37A10S716 GFTFSDYWMD VH CDR1 359 37A10S716NIDESGSITEYSPFVKG VHCDR2 360 37A10S716 WGRFGFDS VH CDR3 361 37A10S716KSSQSLLSGSFNYLT VL CDR1 362 37A10S716 YASTRHT VL CDR2 363 37A10S716HHHYNAPPT VL CDR3 364 37A10S717EVQLVESGGG LVQPGGSLRL SCAASGFTFS DYWMDWVRQA PGKGLVWVSN heavy chainIDESGSITEY SPFVKGRFTI SRDNAKNTLY LQMNSLRAED TAVYYCTRWG variable regionRFGFDSWGQG TLVTVSS 365 37A10S717DIVMTQSPDS LAVSLGERAT INCKSSQSLL SGSFNYLTWY QQKPGQPPKL light chainLIFYASTRET GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCHHHYNAPP variable regionTFGPGTKVDI K 366 37A10S717 GFTFSDYWMD VH CDR1 367 37A10S717NIDESGSITEYSPFVKG VHCDR2 368 37A10S717 WGRFGFDS VH CDR3 369 37A10S717KSSQSLLSGSFNYLT VL CDR1 370 37A10S717 YASTRET VL CDR2 371 37A10S717HHHYNAPPT VL CDR3 372 37A10S718EVQLVESGGG LVQPGGSLRL SCAASGFTFS DYWMDWVRQA PGKGLVWVSN heavy chainIDESGSITEY SPFVKGRFTI SRDNAKNTLY LQMNSLRAED TAVYYCTRWG variable regionRFGFDSWGQG TLVTVSS 373 37A10S718DIVMTQSPDS LAVSLGERAT INCKSSQSLL SGSFNYLTWY QQKPGQPPKL light chainLIFYASTRQT GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCHHHYNAPP variable regionTFGPGTKVDI K 374 37A10S718 GFTFSDYWMD VH CDR1 375 37A10S718NIDESGSITEYSPFVKG VHCDR2 376 37A10S718 WGRFGFDS VH CDR3 377 37A10S718KSSQSLLSGSFNYLT VL CDR1 378 37A10S718 YASTRQT VL CDR2 379 37A10S718HHHYNAPPT VL CDR3 380 37A10S713EVQLVESGGG LVQPGGSLRL SCAASGFTFS DYWMDWVRQA PGKGLVWVSN human IgG1IDEDGSITEY SPFVKGRFTI SRDNAKNTLY LQMNSLRAED TAVYYCTRWG heavy chainRFGFDSWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYFPEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYICNVNHKPSNTK VDKKVEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDTLMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYTLPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDSDGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK 381 37A10S713DIVMTQSPDS LAVSLGERAT INCKSSQSLL SGSFNYLTWY QQKPGQPPKL human κ lightLIFYASTRHT GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCHHHYNAPP chainTFGPGTKVDI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC

1-106. (canceled)
 107. An isolated nucleic acid encoding a monoclonalantibody that binds to human Programmed Death 1 (PD-1), wherein theantibody comprises (a) HCDR1 comprising the amino acid sequence of SEQID NO: 21; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO:22; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO: 23; (d)LCDR1 comprising the amino acid sequence of SEQ ID NO: 25; (e) LCDR2comprising the amino acid sequence of SEQ ID NO: 26; and (f) LCDR3comprising the amino acid sequence of SEQ ID NO:
 27. 108. The isolatednucleic acid of claim 107, wherein the antibody comprises a heavy chainvariable region (V_(H)) that is at least 90% identical to the amino acidsequence of SEQ ID NO: 20 and a light chain variable region (V_(L)) thatis at least 90% identical to the amino acid sequence of SEQ ID NO: 24.109. The isolated nucleic acid of claim 107, wherein the antibodycomprises a V_(H) comprising the amino acid sequence of SEQ ID NO: 20and a V_(L) comprising the amino acid sequence of SEQ ID NO:
 24. 110.The isolated nucleic acid of claim 107, wherein the antibody is a humanantibody or a chimeric antibody.
 111. The isolated nucleic acid of claim107, wherein the antibody is an antibody fragment selected from a Fab,Fab′, Fv, scFv or (Fab′)₂ fragment.
 112. The isolated nucleic acid ofclaim 107, wherein the antibody is a full length antibody.
 113. Theisolated nucleic acid of claim 109, wherein the antibody comprises ahuman IgG4 constant region.
 114. An isolated nucleic acid encoding aheavy chain or a light chain of an anti-PD-1 antibody, wherein (a) theheavy chain comprises (i) HCDR1 comprising the amino acid sequence ofSEQ ID NO: 21; (ii) HCDR2 comprising the amino acid sequence of SEQ IDNO: 22; and (iii) HCDR3 comprising the amino acid sequence of SEQ ID NO:23; and (b) the light chain comprises (i) LCDR1 comprising the aminoacid sequence of SEQ ID NO: 25; (ii) LCDR2 comprising the amino acidsequence of SEQ ID NO: 26; and (iii) LCDR3 comprising the amino acidsequence of SEQ ID NO:
 27. 115. The isolated nucleic acid of claim 114,wherein the heavy chain comprises a heavy chain variable region (V_(H))that is at least 90% identical to the amino acid sequence of SEQ ID NO:20, and the light chain comprises a light chain variable region (V_(L))that is at least 90% identical to the amino acid sequence of SEQ ID NO:24.
 116. The isolated nucleic acid of claim 114, wherein the heavy chaincomprises a V_(H) comprising the amino acid sequence of SEQ ID NO: 20,and the light chain comprises a V_(L) comprising the amino acid sequenceof SEQ ID NO:
 24. 117. The isolated nucleic acid of claim 114, whereinthe antibody is a human antibody or a chimeric antibody.
 118. Theisolated nucleic acid of claim 114, wherein the antibody is a fulllength antibody.
 119. The isolated nucleic acid of claim 116, whereinthe isolated nucleic acid encodes a heavy chain of an anti-PD-1antibody, and wherein the antibody comprises a human IgG4 constantregion.
 120. A vector comprising the nucleic acid of claim
 107. 121. Avector comprising the nucleic acid of claim
 109. 122. A vectorcomprising the nucleic acid of claim
 113. 123. A vector comprising thenucleic acid of claim
 114. 124. A vector comprising the nucleic acid ofclaim
 116. 125. A vector comprising the nucleic acid of claim
 119. 126.A host cell comprising the nucleic acid of claim
 107. 127. A host cellcomprising the nucleic acid of claim
 109. 128. A host cell comprisingthe nucleic acid of claim
 113. 129. A host cell that expresses amonoclonal antibody that binds to human Programmed Death 1 (PD-1),wherein the antibody comprises (a) HCDR1 comprising the amino acidsequence of SEQ ID NO: 21; (b) HCDR2 comprising the amino acid sequenceof SEQ ID NO: 22; (c) HCDR3 comprising the amino acid sequence of SEQ IDNO: 23; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO: 25;(e) LCDR2 comprising the amino acid sequence of SEQ ID NO: 26; and (f)LCDR3 comprising the amino acid sequence of SEQ ID NO:
 27. 130. The hostcell of claim 129, wherein the antibody comprises a heavy chain variableregion (V_(H)) that is at least 90% identical to the amino acid sequenceof SEQ ID NO: 20 and a light chain variable region (V_(L)) that is atleast 90% identical to the amino acid sequence of SEQ ID NO:
 24. 131.The host cell of claim 129, wherein the antibody comprises a V_(H)comprising the amino acid sequence of SEQ ID NO: 20 and a V_(L)comprising the amino acid sequence of SEQ ID NO:
 24. 132. The host cellof claim 129, wherein the antibody is a human antibody or a chimericantibody.
 133. The host cell of claim 129, wherein the antibody is anantibody fragment selected from a Fab, Fab′, Fv, scFv or (Fab′)₂fragment.
 134. The host cell of claim 129, wherein the antibody is afull length antibody.
 135. The host cell of claim 129, wherein theantibody comprises a human IgG4 constant region.
 136. A method formaking an anti-PD-1 antibody, comprising culturing the host cell ofclaim 126 under conditions suitable for expression of the antibody, andisolating the antibody.
 137. A method for making an anti-PD-1 antibody,comprising culturing the host cell of claim 127 under conditionssuitable for expression of the antibody, and isolating the antibody.138. A method for making an anti-PD-1 antibody, comprising culturing thehost cell of claim 128 under conditions suitable for expression of theantibody, and isolating the antibody.
 139. A method for making ananti-PD-1 antibody, comprising culturing the host cell of claim 129under conditions suitable for expression of the antibody, and isolatingthe antibody.
 140. A method for making an anti-PD-1 antibody, comprisingculturing the host cell of claim 131 under conditions suitable forexpression of the antibody, and isolating the antibody.
 141. A methodfor making an anti-PD-1 antibody, comprising culturing the host cell ofclaim 135 under conditions suitable for expression of the antibody, andisolating the antibody.